Structured Review

Roche proteinase k
RCA can detect rSDS-PrP Sc oligomers at early stages of the disease. a Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [ 16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels. b Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above. c To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of <t>proteinase</t> K and processed as described in the legend to Fig. 4a . The asterisk in b and c indicates the position of oligomer traces
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Images

1) Product Images from "A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases"

Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

Journal: Molecular Neurodegeneration

doi: 10.1186/s13024-016-0074-7

RCA can detect rSDS-PrP Sc oligomers at early stages of the disease. a Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [ 16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels. b Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above. c To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of proteinase K and processed as described in the legend to Fig. 4a . The asterisk in b and c indicates the position of oligomer traces
Figure Legend Snippet: RCA can detect rSDS-PrP Sc oligomers at early stages of the disease. a Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [ 16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels. b Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above. c To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of proteinase K and processed as described in the legend to Fig. 4a . The asterisk in b and c indicates the position of oligomer traces

Techniques Used: Infection, Incubation, Western Blot

A MR100-based assay can differentiate between prion-infected and normal brain homogenates without proteinase K digestion. a Schematic description of the RCA protocol to test brain homogenates without PK digestion. Brain tissues were freshly homogenized in microbead-containing tubes. Normal brain homogenates (NBH) or prion-infected brain homogenates (IBH) were incubated with MR100 for 1 h, at room temperature, leading to a precipitation of PrP isoforms. After a short centrifugation step, the pellet with MR100 (orange tube) concentrates PrP isoforms, whereas no pellet is detectable with DMSO. b Comparison of DMSO, P30 and MR100 precipitation capabilities using the RCA protocol. Fifty μL of 10 % 22 L infected brain homogenates were diluted in 300 μL of PBS/2 % sarcosyl and incubated using either 1.5 mM of P30 or MR100 or an equivalent volume of the solvent alone (DMSO), at room temperature for 1 h. Then, samples were centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant was mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with an equal volume of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix according to standard procedures [ 16 ]. The samples (S/P) were analyzed by western blotting using SAF mix anti-PrP antibodies. c Comparison of infected versus non-infected brain homogenates processed with the RCA protocol. Fifty microliters of 10 % freshly homogenized brain tissues from normal (NBH) or 22 L prion-infected (IBH) mice were processed according to the RCA protocol described in A and B. Thirty microliters of supernatant (S) or pellet (P) were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix as described above. Molecular masses (20–75 kDa) are indicated on the left side of the panels
Figure Legend Snippet: A MR100-based assay can differentiate between prion-infected and normal brain homogenates without proteinase K digestion. a Schematic description of the RCA protocol to test brain homogenates without PK digestion. Brain tissues were freshly homogenized in microbead-containing tubes. Normal brain homogenates (NBH) or prion-infected brain homogenates (IBH) were incubated with MR100 for 1 h, at room temperature, leading to a precipitation of PrP isoforms. After a short centrifugation step, the pellet with MR100 (orange tube) concentrates PrP isoforms, whereas no pellet is detectable with DMSO. b Comparison of DMSO, P30 and MR100 precipitation capabilities using the RCA protocol. Fifty μL of 10 % 22 L infected brain homogenates were diluted in 300 μL of PBS/2 % sarcosyl and incubated using either 1.5 mM of P30 or MR100 or an equivalent volume of the solvent alone (DMSO), at room temperature for 1 h. Then, samples were centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant was mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with an equal volume of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix according to standard procedures [ 16 ]. The samples (S/P) were analyzed by western blotting using SAF mix anti-PrP antibodies. c Comparison of infected versus non-infected brain homogenates processed with the RCA protocol. Fifty microliters of 10 % freshly homogenized brain tissues from normal (NBH) or 22 L prion-infected (IBH) mice were processed according to the RCA protocol described in A and B. Thirty microliters of supernatant (S) or pellet (P) were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix as described above. Molecular masses (20–75 kDa) are indicated on the left side of the panels

Techniques Used: Infection, Incubation, Centrifugation, Western Blot, Mouse Assay

rSDS-PrP Sc oligomers are detected in patients with new variant CJD (vCJD) when tested with RCA. a-b Frozen, homogenized brain samples from two patients with vCJD, one patient with sCJD (codon 129 M/M genotype) (positive control) and one healthy control (NBH) were from NIBSC. Each sample was identified by the number attributed by the NIBSC. The RCA assay was carried out as before (see legend to Fig. 5 ) but adapted to human samples: 50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl were incubated with 2 mM MR100 for 2 h. Before centrifugation, 30 μL was collected and mixed with 30 μL of 2X loading buffer for immunoblotting analysis ( a ). The rest of the sample was centrifuged at 11,000 g for 5 min, and supernatants (S) and pellets (P) were immunoblotted with the SAFmix ( b ) [ 16 ]. c-d For comparison, the same brain homogenates (50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl) were processed with the classical proteinase K digestion assay without MR100. Samples were analyzed before ( c ), and after proteinase K digestion (125 μg/mL PK at 37 °C for 1 h) ( d ). The reaction was stopped by addition of a protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix. Molecular masses (20–75 kDa) are on the left side of the panels
Figure Legend Snippet: rSDS-PrP Sc oligomers are detected in patients with new variant CJD (vCJD) when tested with RCA. a-b Frozen, homogenized brain samples from two patients with vCJD, one patient with sCJD (codon 129 M/M genotype) (positive control) and one healthy control (NBH) were from NIBSC. Each sample was identified by the number attributed by the NIBSC. The RCA assay was carried out as before (see legend to Fig. 5 ) but adapted to human samples: 50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl were incubated with 2 mM MR100 for 2 h. Before centrifugation, 30 μL was collected and mixed with 30 μL of 2X loading buffer for immunoblotting analysis ( a ). The rest of the sample was centrifuged at 11,000 g for 5 min, and supernatants (S) and pellets (P) were immunoblotted with the SAFmix ( b ) [ 16 ]. c-d For comparison, the same brain homogenates (50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl) were processed with the classical proteinase K digestion assay without MR100. Samples were analyzed before ( c ), and after proteinase K digestion (125 μg/mL PK at 37 °C for 1 h) ( d ). The reaction was stopped by addition of a protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix. Molecular masses (20–75 kDa) are on the left side of the panels

Techniques Used: Variant Assay, Positive Control, Incubation, Centrifugation, Protease Inhibitor, Western Blot

MR100 has a stronger PrP Sc oligomer-inducing activity in prion-infected N2a58/22 L cells than P30 and A6. a Effect of the newly synthesized MR1, MR2 and MR100 compounds in prion-infected N2a58/22 L cells. Cells were left untreated (CTR) or incubated with 20 μM of A6 (positive control), MR1 and MR2 (synthesis intermediates), MR100, or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix (a mixture of the anti-PrP SAF60, SAF69 and SAF70 monoclonal antibodies) after proteinase K (PK) digestion. b Comparison of the oligomer-inducing activity of P30, A6 and MR100. Prion-infected N2a58/22 L cells were incubated with 0.5, 1 or 2.5 μM of each compound, or 20 μL DMSO (DM) for 4 days. Protein lysates were then analyzed by immunoblotting with the SAF mix after PK digestion. c MR100 dose-response curve in prion-infected N2a58/22 L cells. Successive dilutions of MR100 in DMSO were used to obtain final concentrations ranging from 10 -12 M (1pM) to 10 -5 M (10 μM). Cells were incubated for 4 days and at confluence they were lysed. Protein lysates were analyzed by immunoblotting with the SAF mix after PK digestion according to the previously described protocol [ 16 , 30 ]. Loading control was performed with antibodies against glyceraldehyde-3-P dehydrogenase (G3PDH) and before proteinase K digestion. Molecular masses (20–50 kDa) are indicated on the left side of the panels
Figure Legend Snippet: MR100 has a stronger PrP Sc oligomer-inducing activity in prion-infected N2a58/22 L cells than P30 and A6. a Effect of the newly synthesized MR1, MR2 and MR100 compounds in prion-infected N2a58/22 L cells. Cells were left untreated (CTR) or incubated with 20 μM of A6 (positive control), MR1 and MR2 (synthesis intermediates), MR100, or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix (a mixture of the anti-PrP SAF60, SAF69 and SAF70 monoclonal antibodies) after proteinase K (PK) digestion. b Comparison of the oligomer-inducing activity of P30, A6 and MR100. Prion-infected N2a58/22 L cells were incubated with 0.5, 1 or 2.5 μM of each compound, or 20 μL DMSO (DM) for 4 days. Protein lysates were then analyzed by immunoblotting with the SAF mix after PK digestion. c MR100 dose-response curve in prion-infected N2a58/22 L cells. Successive dilutions of MR100 in DMSO were used to obtain final concentrations ranging from 10 -12 M (1pM) to 10 -5 M (10 μM). Cells were incubated for 4 days and at confluence they were lysed. Protein lysates were analyzed by immunoblotting with the SAF mix after PK digestion according to the previously described protocol [ 16 , 30 ]. Loading control was performed with antibodies against glyceraldehyde-3-P dehydrogenase (G3PDH) and before proteinase K digestion. Molecular masses (20–50 kDa) are indicated on the left side of the panels

Techniques Used: Activity Assay, Infection, Synthesized, Incubation, Positive Control

MR100 did not induce SDS resistant PrP C oligomers. a Parental non-infected cells, N2a58, were left untreated (CTR) or incubated with 20 μM of MR100 or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix before or after PK digestion. b N2a58 cell lines were left untreated (CTR) or incubated with various concentration of MR100 from 5 to 40 μM or 40 μL of DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF 32 before or after PK digestion. Loading control was performed with antibodies against β actin and before proteinase K digestion. c Schematic representation of the protocols used to test if PrP C isoforms are part of rSDS-oligomers (Left panel). First step, N2a58/22 L lysates were incubated with 20 μM of MR100 or with proteinase K to eliminate PrP C , then in the second step, MR100-exposed lysates were digested with proteinase K, while proteinase K digested samples were incubated with MR100. PrP Sc species were then analyzed by western blotting. Western blot analysis of the samples processed according to the two different protocols using the SAFmix of anti-PrP antibodies (Right panel). CTR, untreated samples, digested by proteinase K; DM, DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels
Figure Legend Snippet: MR100 did not induce SDS resistant PrP C oligomers. a Parental non-infected cells, N2a58, were left untreated (CTR) or incubated with 20 μM of MR100 or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix before or after PK digestion. b N2a58 cell lines were left untreated (CTR) or incubated with various concentration of MR100 from 5 to 40 μM or 40 μL of DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF 32 before or after PK digestion. Loading control was performed with antibodies against β actin and before proteinase K digestion. c Schematic representation of the protocols used to test if PrP C isoforms are part of rSDS-oligomers (Left panel). First step, N2a58/22 L lysates were incubated with 20 μM of MR100 or with proteinase K to eliminate PrP C , then in the second step, MR100-exposed lysates were digested with proteinase K, while proteinase K digested samples were incubated with MR100. PrP Sc species were then analyzed by western blotting. Western blot analysis of the samples processed according to the two different protocols using the SAFmix of anti-PrP antibodies (Right panel). CTR, untreated samples, digested by proteinase K; DM, DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

Techniques Used: Infection, Incubation, Concentration Assay, Western Blot

MR100 shows oligomer-inducing activity in brain homogenates from prion-infected rodents. a MR100 oligomer-inducing activity was tested using freshly homogenized rodent brain tissues infected with the 22 L (mice) or the 263 K prion strain (hamsters). Fifty μL of 10 % mouse or hamster brain homogenates were diluted in 300 μL PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 (corresponding to 150 μL of 5 mM MR100) at room temperature for 1 h or with 150 μL of DMSO as control, and then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins). PK digestion was stopped by addition of a cocktail of protease inhibitors (Complete), before analysis of rPrP Sc by western blotting with the SAF mix according to the previously described protocol [ 16 ]. CTR: untreated 22 L- or 263 K-infected brain homogenates (negative control); DM: 22 L- or 263 K-infected brain homogenates incubated with DMSO. b Comparison of P30 and MR100 oligomer-inducing activity on the 22 L prion strain, before and after proteinase K digestion. Fifty μL of 10 % 22 L-infected brain homogenates were diluted in 350 μL PBS/2 % Sarkosyl, incubated with 1 mM MR100 or P30 (corresponding to 100 μL of 5 mM MR100 or P30), at room temperature for 1 h. Then, aliquots of 30 μL were taken before addition of proteinase K, to perform western blot (PK-) probed with SAF mix, but also with anti-β-actin antibodies as loading controls. The rest of the sample was then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins) (PK+). The reaction was stopped by addition of the protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix as in A. DM: 22 L-infected brain homogenates incubated with 100 μL DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels
Figure Legend Snippet: MR100 shows oligomer-inducing activity in brain homogenates from prion-infected rodents. a MR100 oligomer-inducing activity was tested using freshly homogenized rodent brain tissues infected with the 22 L (mice) or the 263 K prion strain (hamsters). Fifty μL of 10 % mouse or hamster brain homogenates were diluted in 300 μL PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 (corresponding to 150 μL of 5 mM MR100) at room temperature for 1 h or with 150 μL of DMSO as control, and then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins). PK digestion was stopped by addition of a cocktail of protease inhibitors (Complete), before analysis of rPrP Sc by western blotting with the SAF mix according to the previously described protocol [ 16 ]. CTR: untreated 22 L- or 263 K-infected brain homogenates (negative control); DM: 22 L- or 263 K-infected brain homogenates incubated with DMSO. b Comparison of P30 and MR100 oligomer-inducing activity on the 22 L prion strain, before and after proteinase K digestion. Fifty μL of 10 % 22 L-infected brain homogenates were diluted in 350 μL PBS/2 % Sarkosyl, incubated with 1 mM MR100 or P30 (corresponding to 100 μL of 5 mM MR100 or P30), at room temperature for 1 h. Then, aliquots of 30 μL were taken before addition of proteinase K, to perform western blot (PK-) probed with SAF mix, but also with anti-β-actin antibodies as loading controls. The rest of the sample was then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins) (PK+). The reaction was stopped by addition of the protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix as in A. DM: 22 L-infected brain homogenates incubated with 100 μL DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

Techniques Used: Activity Assay, Infection, Mouse Assay, Incubation, Western Blot, Negative Control, Protease Inhibitor

2) Product Images from "Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism"

Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002128

Effect of accessory cofactors on the propagation of ΔPBD PrP Sc  molecules. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc  molecules were propagated by sPMCA with autologous PrP C  prepared from Chinese hamster ovary (CHO) cells. One set of reactions was supplemented with  Prnp 0/0  mouse brain homogenate (+cofactor), while a second set received only buffer (−cofactor). One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. –PK reactions for ΔC-PBD and ΔΔ-PBD were loaded with ¼ volume. PrP was detected by immunoblot (anti-PrP 27/33).
Figure Legend Snippet: Effect of accessory cofactors on the propagation of ΔPBD PrP Sc molecules. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc molecules were propagated by sPMCA with autologous PrP C prepared from Chinese hamster ovary (CHO) cells. One set of reactions was supplemented with Prnp 0/0 mouse brain homogenate (+cofactor), while a second set received only buffer (−cofactor). One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. –PK reactions for ΔC-PBD and ΔΔ-PBD were loaded with ¼ volume. PrP was detected by immunoblot (anti-PrP 27/33).

Techniques Used: In Vitro, Generated

Biochemical and neuropathological analysis of mice inoculated with in vitro -generated PrP Sc molecules. Brains were dissected from wild-type mice showing terminal scrapie signs (PrP Sc and ΔC-PBD PrP Sc inocula) or similarly aged mice not displaying scrapie signs (mock-propagated, ΔN-PBD PrP Sc , and ΔΔ-PBD PrP Sc inocula). ( A ) Equivalent amounts of 10% brain homogenate were treated with buffer (−PK) or 25 µg/mL proteinase K (+PK to show PrP Sc ) and detected by anti-PrP (6D11) immunoblot. A greater exposure of the same immunoblot is displayed below, to illustrate samples containing low amounts of PrP Sc . ( B ) Neuropathology of cerebellum and hippocampus. Brain sections were stained with hematoxylin and eosin (H E). The black bar denotes 100 µm.
Figure Legend Snippet: Biochemical and neuropathological analysis of mice inoculated with in vitro -generated PrP Sc molecules. Brains were dissected from wild-type mice showing terminal scrapie signs (PrP Sc and ΔC-PBD PrP Sc inocula) or similarly aged mice not displaying scrapie signs (mock-propagated, ΔN-PBD PrP Sc , and ΔΔ-PBD PrP Sc inocula). ( A ) Equivalent amounts of 10% brain homogenate were treated with buffer (−PK) or 25 µg/mL proteinase K (+PK to show PrP Sc ) and detected by anti-PrP (6D11) immunoblot. A greater exposure of the same immunoblot is displayed below, to illustrate samples containing low amounts of PrP Sc . ( B ) Neuropathology of cerebellum and hippocampus. Brain sections were stained with hematoxylin and eosin (H E). The black bar denotes 100 µm.

Techniques Used: Mouse Assay, In Vitro, Generated, Staining

Interaction of ΔC-PBD PrP Sc with mutant PrP molecules. ( A ) Binding of ΔC-PBD PrP Sc to PrP. ΔC-PBD PrP Sc was incubated with wild-type or polybasic mutant (ΔC-PBD, ΔN-PBD) myc-tagged PrP. Bound PrP Sc was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (27/33) immunoblot. ( B ) Propagation of ΔC-PBD PrP Sc . ΔC-PBD PrP Sc was propagated by sPMCA with polybasic deletion mutant PrP C prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with Prnp 0/0 mouse brain homogenate. One sample of each reaction was not subjected to protease digestion (−PK), loading ¼ of volume. All others were subjected to limited proteolysis by 25 µg/mL proteinase K digestion. PrP was detected by immunoblot (anti-PrP 27/33).
Figure Legend Snippet: Interaction of ΔC-PBD PrP Sc with mutant PrP molecules. ( A ) Binding of ΔC-PBD PrP Sc to PrP. ΔC-PBD PrP Sc was incubated with wild-type or polybasic mutant (ΔC-PBD, ΔN-PBD) myc-tagged PrP. Bound PrP Sc was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (27/33) immunoblot. ( B ) Propagation of ΔC-PBD PrP Sc . ΔC-PBD PrP Sc was propagated by sPMCA with polybasic deletion mutant PrP C prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with Prnp 0/0 mouse brain homogenate. One sample of each reaction was not subjected to protease digestion (−PK), loading ¼ of volume. All others were subjected to limited proteolysis by 25 µg/mL proteinase K digestion. PrP was detected by immunoblot (anti-PrP 27/33).

Techniques Used: Mutagenesis, Binding Assay, Incubation

ΔPBD PrP Sc  molecules seeding wild-type brain homogenate sPMCA reactions. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc  molecules were propagated by sPMCA for three rounds with wild-type mouse brain homogenate, containing wild-type PrP C  substrate. Control reactions seeded with wild-type native RML prions and unseeded reactions were also tested. One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 6D11).
Figure Legend Snippet: ΔPBD PrP Sc molecules seeding wild-type brain homogenate sPMCA reactions. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc molecules were propagated by sPMCA for three rounds with wild-type mouse brain homogenate, containing wild-type PrP C substrate. Control reactions seeded with wild-type native RML prions and unseeded reactions were also tested. One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 6D11).

Techniques Used: In Vitro, Generated

Interaction of wild-type PrP Sc with mutant PrP molecules. ( A ) Binding of PrP Sc to PrP. RML scrapie-infected mouse brain homogenate was incubated with myc-tagged PrP of wild-type sequence or lacking the central (ΔC-PBD: Δ100–109) or N-terminal (ΔN-PBD: Δ23–28) polybasic domain. Bound PrP Sc was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (6D11) immunoblot. ( B ) Propagation of PrP Sc . RML scrapie-infected mouse brain homogenate was propagated by serial protein misfolding cyclic amplification (sPMCA) for five rounds with wild-type or polybasic deletion mutant PrP C prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with Prnp 0/0 mouse brain homogenate. In addition to scrapie-seeded reactions, an unseeded reaction was performed with ΔC-PBD PrP substrate. One sample of each reaction was not subjected to protease digestion (−PK). All others were subjected to limited proteolysis with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 27/33).
Figure Legend Snippet: Interaction of wild-type PrP Sc with mutant PrP molecules. ( A ) Binding of PrP Sc to PrP. RML scrapie-infected mouse brain homogenate was incubated with myc-tagged PrP of wild-type sequence or lacking the central (ΔC-PBD: Δ100–109) or N-terminal (ΔN-PBD: Δ23–28) polybasic domain. Bound PrP Sc was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (6D11) immunoblot. ( B ) Propagation of PrP Sc . RML scrapie-infected mouse brain homogenate was propagated by serial protein misfolding cyclic amplification (sPMCA) for five rounds with wild-type or polybasic deletion mutant PrP C prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with Prnp 0/0 mouse brain homogenate. In addition to scrapie-seeded reactions, an unseeded reaction was performed with ΔC-PBD PrP substrate. One sample of each reaction was not subjected to protease digestion (−PK). All others were subjected to limited proteolysis with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 27/33).

Techniques Used: Mutagenesis, Binding Assay, Infection, Incubation, Sequencing, Protein Misfolding Cyclic Amplification

3) Product Images from "Detection of protease-resistant cervid prion protein in water from a CWD-endemic area"

Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

Journal: Prion

doi:

PrP CWD  amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD  from five replicate
Figure Legend Snippet: PrP CWD amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD from five replicate

Techniques Used: Amplification

Effects of flocculation and alum on PrP CWD  amplification. All samples were digested with Proteinase K except lane 1. (A) PrP CWD  precipitates with flocculant water sample. Lanes 1 and 2 shows amplified NBH control. Lanes 3–6 show amplified samples
Figure Legend Snippet: Effects of flocculation and alum on PrP CWD amplification. All samples were digested with Proteinase K except lane 1. (A) PrP CWD precipitates with flocculant water sample. Lanes 1 and 2 shows amplified NBH control. Lanes 3–6 show amplified samples

Techniques Used: Flocculation, Amplification

PrP CWD  detection limit in water. (A) All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lanes 2 through 12 show amplified samples at the indicated starting dilution of CWD-positive brain into water. Lanes 13 and
Figure Legend Snippet: PrP CWD detection limit in water. (A) All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lanes 2 through 12 show amplified samples at the indicated starting dilution of CWD-positive brain into water. Lanes 13 and

Techniques Used: Amplification

PrP CWD  detected in raw water samples collected at a time of increased snow-melt runoff. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. Lanes 3–8 show Horsetooth reservoir
Figure Legend Snippet: PrP CWD detected in raw water samples collected at a time of increased snow-melt runoff. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. Lanes 3–8 show Horsetooth reservoir

Techniques Used: Amplification

Abrogation of PrP CWD  amplification from 5-22-07 raw PR water samples by PMCA using murine PrP C  substrate or Proteinase K pre-treatment. All western blot samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows
Figure Legend Snippet: Abrogation of PrP CWD amplification from 5-22-07 raw PR water samples by PMCA using murine PrP C substrate or Proteinase K pre-treatment. All western blot samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows

Techniques Used: Amplification, Western Blot

Normal brain homogenate negative controls. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. NBH, Normal brain homogenate control. +, 1:100,000 positive amplification control. Molecular weight markers in kilodaltons
Figure Legend Snippet: Normal brain homogenate negative controls. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. NBH, Normal brain homogenate control. +, 1:100,000 positive amplification control. Molecular weight markers in kilodaltons

Techniques Used: Amplification, Molecular Weight

4) Product Images from "Rabbits are not resistant to prion infection"

Article Title: Rabbits are not resistant to prion infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1120076109

Electrophoretic patterns of rabbit and rabbit PrP over-expressing transgenic (raPrPTg) mouse adapted de novo RaTgPrP Sc strains. ( A ) Brain homogenate from de novo RaPrP Sc -inoculated rabbit (lanes 1 and 5) was compared biochemically with three brain homogenates from de novo RaPrP Sc -inoculated raPrPTg mice (coded as 01, 02, and 03). Both types of samples [rabbit (first passage) and raPrPTg (second passage)–derived prion strains] were indistinguishable according to the electrophoretic migration pattern and glycosylation profile, suggesting that de novo RaPrP Sc strain was stable after its transmission in a rabbit PrP transgenic model. ( B ) Brain homogenate from de novo RaPrP Sc -inoculated rabbit (first passage, lane 1) was compared biochemically with two brain homogenates from de novo RaPrP Sc -inoculated rabbits (second passage, lanes 2–5). Two different areas [frontal cortex (FC) and midbrain (MD)] from two animals (477 dpi, lanes 2–3 ; 540 dpi, lanes 4–5) were analyzed. Both types of samples [rabbit (first passage) and rabbit (second passage)–derived prion strains] were indistinguishable according to the electrophoretic migration pattern and glycosylation profile, suggesting that de novo RaPrP Sc strain was stable after its transmission in rabbits. Samples were digested with 100 μg/mL of proteinase K and were analyzed by Western blot using monoclonal antibodies L42 ( Left ) and SAF84 ( Right ). Control, normal rabbit brain homogenate.
Figure Legend Snippet: Electrophoretic patterns of rabbit and rabbit PrP over-expressing transgenic (raPrPTg) mouse adapted de novo RaTgPrP Sc strains. ( A ) Brain homogenate from de novo RaPrP Sc -inoculated rabbit (lanes 1 and 5) was compared biochemically with three brain homogenates from de novo RaPrP Sc -inoculated raPrPTg mice (coded as 01, 02, and 03). Both types of samples [rabbit (first passage) and raPrPTg (second passage)–derived prion strains] were indistinguishable according to the electrophoretic migration pattern and glycosylation profile, suggesting that de novo RaPrP Sc strain was stable after its transmission in a rabbit PrP transgenic model. ( B ) Brain homogenate from de novo RaPrP Sc -inoculated rabbit (first passage, lane 1) was compared biochemically with two brain homogenates from de novo RaPrP Sc -inoculated rabbits (second passage, lanes 2–5). Two different areas [frontal cortex (FC) and midbrain (MD)] from two animals (477 dpi, lanes 2–3 ; 540 dpi, lanes 4–5) were analyzed. Both types of samples [rabbit (first passage) and rabbit (second passage)–derived prion strains] were indistinguishable according to the electrophoretic migration pattern and glycosylation profile, suggesting that de novo RaPrP Sc strain was stable after its transmission in rabbits. Samples were digested with 100 μg/mL of proteinase K and were analyzed by Western blot using monoclonal antibodies L42 ( Left ) and SAF84 ( Right ). Control, normal rabbit brain homogenate.

Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Derivative Assay, Migration, Transmission Assay, Western Blot

Comparative electrophoretic patterns from different prion strains. ( A ) Brain homogenate from de novo RaPrP Sc inoculated rabbit was compared biochemically with the most typical prion strains from different species. To have a clear reference and accurate comparison of the different electrophoretic migration patterns, de novo RaPrP Sc was run three times (arrows). De novo RaPrP Sc shows unique biochemical characteristics determined by electrophoretic migration pattern and glycosylation profile in that the unglycosylated band migrates further compared with all other strains with the exception of sporadic Creutzfeldt-Jakob disease (sCJD). However, sCJD can be easily distinguished from the RaPrP Sc strain because of its glycosylation profile and the specific migration of the di-glycosylation band, which does not migrate as far. ( B ) In vitro generated de novo and ME7 RaPrP res (lanes 1 and 2, respectively) are biochemically compared with de novo RaPrP Sc generated in vivo (lane 3). Lane 4 shows the biochemical pattern of RaPrP res generated in vitro by PMCA, using in vivo de novo RaPrP Sc as seed. Although not identical, the in vivo pattern (lane 3) was very similar to that previously observed in the material generated in vitro (lanes 1, 2, and 4). All samples were digested with 100 μg/mL of proteinase K and were analyzed by Western blot using monoclonal antibody D18. Curved arrows indicate in vitro passage by PMCA. Control, normal rabbit brain homogenate.
Figure Legend Snippet: Comparative electrophoretic patterns from different prion strains. ( A ) Brain homogenate from de novo RaPrP Sc inoculated rabbit was compared biochemically with the most typical prion strains from different species. To have a clear reference and accurate comparison of the different electrophoretic migration patterns, de novo RaPrP Sc was run three times (arrows). De novo RaPrP Sc shows unique biochemical characteristics determined by electrophoretic migration pattern and glycosylation profile in that the unglycosylated band migrates further compared with all other strains with the exception of sporadic Creutzfeldt-Jakob disease (sCJD). However, sCJD can be easily distinguished from the RaPrP Sc strain because of its glycosylation profile and the specific migration of the di-glycosylation band, which does not migrate as far. ( B ) In vitro generated de novo and ME7 RaPrP res (lanes 1 and 2, respectively) are biochemically compared with de novo RaPrP Sc generated in vivo (lane 3). Lane 4 shows the biochemical pattern of RaPrP res generated in vitro by PMCA, using in vivo de novo RaPrP Sc as seed. Although not identical, the in vivo pattern (lane 3) was very similar to that previously observed in the material generated in vitro (lanes 1, 2, and 4). All samples were digested with 100 μg/mL of proteinase K and were analyzed by Western blot using monoclonal antibody D18. Curved arrows indicate in vitro passage by PMCA. Control, normal rabbit brain homogenate.

Techniques Used: Migration, In Vitro, Generated, In Vivo, Western Blot

Biochemical analysis of different rabbit PrP res (RaPrP res ) generated in vitro by serial automated PMCA (saPMCA) using a New Zealand White rabbit brain homogenate as substrate. Rabbit brain homogenates seeded with different prion strains (mouse: RML, ME7, 22L, 139A, 79A and 22F, mule deer: CWD, cattle: BSE, sheep: SSPB/1 and CH1641) or unseeded (de novo) were subjected to saPMCA. Seeded samples from round 10 and the unseeded sample from round 20 were digested with 100 μg/mL of proteinase K (PK) and analyzed by Western blot using monoclonal antibody D18. Three differential electrophoretic migration patterns are shown, depending on the seed that was used: ( i ) the pattern for the unglycosylated band that migrated the farthest (18–19 K d , sheep and mouse adapted scrapie strains); ( ii ) the intermediate migration pattern for the same band (19–20 K d , BSE); and ( iii ) the migration pattern for the unglycosylated band that migrated the least (20-21 K d , CWD). RML and ME7 RaPrP res samples are used in all blots as reference for determining the different electrophoretic migration patterns. Control, normal rabbit brain homogenate.
Figure Legend Snippet: Biochemical analysis of different rabbit PrP res (RaPrP res ) generated in vitro by serial automated PMCA (saPMCA) using a New Zealand White rabbit brain homogenate as substrate. Rabbit brain homogenates seeded with different prion strains (mouse: RML, ME7, 22L, 139A, 79A and 22F, mule deer: CWD, cattle: BSE, sheep: SSPB/1 and CH1641) or unseeded (de novo) were subjected to saPMCA. Seeded samples from round 10 and the unseeded sample from round 20 were digested with 100 μg/mL of proteinase K (PK) and analyzed by Western blot using monoclonal antibody D18. Three differential electrophoretic migration patterns are shown, depending on the seed that was used: ( i ) the pattern for the unglycosylated band that migrated the farthest (18–19 K d , sheep and mouse adapted scrapie strains); ( ii ) the intermediate migration pattern for the same band (19–20 K d , BSE); and ( iii ) the migration pattern for the unglycosylated band that migrated the least (20-21 K d , CWD). RML and ME7 RaPrP res samples are used in all blots as reference for determining the different electrophoretic migration patterns. Control, normal rabbit brain homogenate.

Techniques Used: Generated, In Vitro, Western Blot, Migration

Proteinase K and guanidine denaturation studies using an in vivo rabbit prion strain. ( A ) De novo RaPrP Sc inoculated rabbit brain homogenate was subjected to several different PK concentrations (80–16,000 μg/mL) at 42 °C for 1 h. ME7 and RML, two well-characterized prion strains, were used as reference. Although the mouse adapted scrapie strains showed a similar PK resistance, the rabbit prion strain showed a much higher resistance to PK treatment. ( B ) De novo RaPrP Sc inoculated rabbit brain homogenate was denatured with different guanidine (GdHCl) concentrations (1.5–4 mol/L) and then subjected to standard Proteinase K (PK) digestion. ME7 and RML prion strains were used as reference. The rabbit prion showed a greater resistance to guanidine denaturation compared with RML and ME7, which had a low or intermediate resistance respectively. Graphs represent an evaluation of Western blots by densitometric analysis from three independent experiments (mean ± SE). All samples were analyzed by Western blot using monoclonal antibody D18.
Figure Legend Snippet: Proteinase K and guanidine denaturation studies using an in vivo rabbit prion strain. ( A ) De novo RaPrP Sc inoculated rabbit brain homogenate was subjected to several different PK concentrations (80–16,000 μg/mL) at 42 °C for 1 h. ME7 and RML, two well-characterized prion strains, were used as reference. Although the mouse adapted scrapie strains showed a similar PK resistance, the rabbit prion strain showed a much higher resistance to PK treatment. ( B ) De novo RaPrP Sc inoculated rabbit brain homogenate was denatured with different guanidine (GdHCl) concentrations (1.5–4 mol/L) and then subjected to standard Proteinase K (PK) digestion. ME7 and RML prion strains were used as reference. The rabbit prion showed a greater resistance to guanidine denaturation compared with RML and ME7, which had a low or intermediate resistance respectively. Graphs represent an evaluation of Western blots by densitometric analysis from three independent experiments (mean ± SE). All samples were analyzed by Western blot using monoclonal antibody D18.

Techniques Used: In Vivo, Western Blot

5) Product Images from "Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice"

Article Title: Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001257

Intranasal prion transmission is independent of lymphotoxin signaling. C57BL/6 mice treated with LTβR-Ig ( A ) or control muIgG ( B ), and mice lacking various components of the LT/TNF system ( D–F , as indicated) were intranasally inoculated with 4×10 5 LD 50 scrapie prions. Survival curves ( A, B, D, E and G ) and respective Western blots ( C, F and H ) indicate efficient prion infection and neuroinvasion. One animal that died early after intranasal inoculation (40 dpi) is reported as intercurrent death (i.d.) for reasons other than scrapie. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends used are as in Fig. 1H .
Figure Legend Snippet: Intranasal prion transmission is independent of lymphotoxin signaling. C57BL/6 mice treated with LTβR-Ig ( A ) or control muIgG ( B ), and mice lacking various components of the LT/TNF system ( D–F , as indicated) were intranasally inoculated with 4×10 5 LD 50 scrapie prions. Survival curves ( A, B, D, E and G ) and respective Western blots ( C, F and H ) indicate efficient prion infection and neuroinvasion. One animal that died early after intranasal inoculation (40 dpi) is reported as intercurrent death (i.d.) for reasons other than scrapie. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends used are as in Fig. 1H .

Techniques Used: Transmission Assay, Mouse Assay, Western Blot, Infection

Intranasal prion transmission in immnunodeficient mice. All mice were intranasally inoculated with 3×10 5 LD 50 prions. ( A ) C1q a −/− mice intranasally inoculated and ( B ) CD21 −/− mice intranasally inoculated are shown. Survival curves illustrate survival after intranasal prion challenge. Respective Western blots of C1qa −/− mice intranasally inoculated ( C, left panel ) and of CD21 −/− mice intranasally inoculated ( C, right panel ) are shown. Survival curves of CXCR5 −/− mice intranasally inoculated are shown ( D ). Respective Western blots of CXCR5 −/− mice intranasally inoculated. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends are as in Fig. 5 .
Figure Legend Snippet: Intranasal prion transmission in immnunodeficient mice. All mice were intranasally inoculated with 3×10 5 LD 50 prions. ( A ) C1q a −/− mice intranasally inoculated and ( B ) CD21 −/− mice intranasally inoculated are shown. Survival curves illustrate survival after intranasal prion challenge. Respective Western blots of C1qa −/− mice intranasally inoculated ( C, left panel ) and of CD21 −/− mice intranasally inoculated ( C, right panel ) are shown. Survival curves of CXCR5 −/− mice intranasally inoculated are shown ( D ). Respective Western blots of CXCR5 −/− mice intranasally inoculated. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends are as in Fig. 5 .

Techniques Used: Transmission Assay, Mouse Assay, Western Blot

Prion transmission by intranasal instillation. ( A ) Rag1 −/− mice intranasally inoculated with RML6 0.1%, ( B ) C57BL/6 mice that have been intranasally inoculated with 3×10 5 LD 50 prions. ( C ) Rag1 −/− mice i.c. inoculated with 3×10 5 LD 50 , ( D ) γ C Rag2 −/− mice intranasally inoculated with 4×10 5 LD 50 or ( E ) Balb/c mice intranasally inoculated with 4×10 5 LD 50 scrapie prions are shown. Survival curves ( A–D ) and respective Western blots ( F–G ) are indicative of efficient prion neuroinvasion. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Brain homogenates derived from a terminally scrapie-sick and a healthy C57BL/6 mouse served as positive and negative controls (s: sick; h: healthy), respectively. Molecular weights (kDa) are indicated on the left side of the blots. ( H and I ) Histopathological lesion severity score described as radar blot (astrogliosis, spongiform change and PrP Sc deposition) in 5 brain regions of both mouse lines exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter.
Figure Legend Snippet: Prion transmission by intranasal instillation. ( A ) Rag1 −/− mice intranasally inoculated with RML6 0.1%, ( B ) C57BL/6 mice that have been intranasally inoculated with 3×10 5 LD 50 prions. ( C ) Rag1 −/− mice i.c. inoculated with 3×10 5 LD 50 , ( D ) γ C Rag2 −/− mice intranasally inoculated with 4×10 5 LD 50 or ( E ) Balb/c mice intranasally inoculated with 4×10 5 LD 50 scrapie prions are shown. Survival curves ( A–D ) and respective Western blots ( F–G ) are indicative of efficient prion neuroinvasion. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Brain homogenates derived from a terminally scrapie-sick and a healthy C57BL/6 mouse served as positive and negative controls (s: sick; h: healthy), respectively. Molecular weights (kDa) are indicated on the left side of the blots. ( H and I ) Histopathological lesion severity score described as radar blot (astrogliosis, spongiform change and PrP Sc deposition) in 5 brain regions of both mouse lines exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter.

Techniques Used: Transmission Assay, Mouse Assay, Western Blot, Derivative Assay

PrP Sc deposition in brains of mice infected with prion aerosols and profiling of NSE-PrP mice. ( A ) Western blot analysis of brain homogenates (10%) from terminal or subclinical tg a 20 mice exposed to aerosols from 20% or 0.1% IBH for 10 min. PK+ or −: with or without proteinase K digest; kDa: Kilo Dalton. ( B–C ): Western blot analyses of brain homogenates from tg a 20 ( B ) or CD1 ( C ) mice exposed to prion aerosols from 20% IBH. ( D ) Histoblot analysis of brains from mice exposed to prion aerosols. Brains of tg a 20 mice challenged with aerosolized 10% (middle panel) or 20% (right panel) IBH showed deposits of PrP Sc in the cortex and mesencephalon. Because the brain of a Prnp o/o mouse showed no signal (left panel), we deduce that the signal in the middle and right panels represents local prion replication. ( E ) Histopathological lesion severity score analysis of 5 brain regions depicted as radar plots [51] (astrogliosis, spongiform change and PrP Sc deposition) derived from tg a 20 , CD1, C57BL/6 and 129SvxC57BL/6 mice exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. ( F ) Histopathological lesion severity score of 5 brain regions shown as radar blot (astrogliosis, spongiform change and PrP Sc deposition) of i.c. prion inoculated tg a 20 , CD1, C57BL/6 and 129SvxC57BL/6 mice. (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. ( G ) Survival curve and ( H ) lesion severity scores of NSE-PrP mice exposed to a 20% aerosolized IBH for 10 min. ( I ) Histological and immunohistochemical characterization of scrapie-affected hippocampi of NSE-PrP mice after exposure to aerosolized 20% IBH. Stain legend as in Fig. 1H . Scale bar: 100µm.
Figure Legend Snippet: PrP Sc deposition in brains of mice infected with prion aerosols and profiling of NSE-PrP mice. ( A ) Western blot analysis of brain homogenates (10%) from terminal or subclinical tg a 20 mice exposed to aerosols from 20% or 0.1% IBH for 10 min. PK+ or −: with or without proteinase K digest; kDa: Kilo Dalton. ( B–C ): Western blot analyses of brain homogenates from tg a 20 ( B ) or CD1 ( C ) mice exposed to prion aerosols from 20% IBH. ( D ) Histoblot analysis of brains from mice exposed to prion aerosols. Brains of tg a 20 mice challenged with aerosolized 10% (middle panel) or 20% (right panel) IBH showed deposits of PrP Sc in the cortex and mesencephalon. Because the brain of a Prnp o/o mouse showed no signal (left panel), we deduce that the signal in the middle and right panels represents local prion replication. ( E ) Histopathological lesion severity score analysis of 5 brain regions depicted as radar plots [51] (astrogliosis, spongiform change and PrP Sc deposition) derived from tg a 20 , CD1, C57BL/6 and 129SvxC57BL/6 mice exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. ( F ) Histopathological lesion severity score of 5 brain regions shown as radar blot (astrogliosis, spongiform change and PrP Sc deposition) of i.c. prion inoculated tg a 20 , CD1, C57BL/6 and 129SvxC57BL/6 mice. (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. ( G ) Survival curve and ( H ) lesion severity scores of NSE-PrP mice exposed to a 20% aerosolized IBH for 10 min. ( I ) Histological and immunohistochemical characterization of scrapie-affected hippocampi of NSE-PrP mice after exposure to aerosolized 20% IBH. Stain legend as in Fig. 1H . Scale bar: 100µm.

Techniques Used: Mouse Assay, Infection, Western Blot, Derivative Assay, Immunohistochemistry, Staining

6) Product Images from "Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome"

Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0190177

Characterization of RiCF. (A)  Representative radiogram showing RNase dose dependent chromosomal fragmentation in AB1157. Plugs were made with 0, 2, 10, 25, 50 or 100 μg RNase and lysed and electrophoresed under standard conditions. CZ, compression zone.  (B)  Quantification showing increase in chromosomal fragmentation in RNase dose-dependent manner. Data points are means of six independent assays ± SEM.  (C)  RNase-effect is not seen in the pre-lyzed cells. Plugs from AB1157 culture were made in the presence of proteinase K, but without any RNase. After overnight lysis and extensive washing, the plugs were incubated with 0, 2, 20 and 100 μg RNase or 100 U of EcoRI for 15 H at 37°C before PFGE.  (D)  Quantification of chromosomal fragmentation showing extreme sensitivity of chromosomes to EcoRI, but not RNase, when plugs were treated with the enzymes after lysis of cells. The experiment is done twice and a representative result is presented.  (E)  A representative radiogram showing kinetics of RiCF. Multiple plugs were made in the presence of RNase (50 μg/plug) and incubated at 62°C for 10, 30, 60, 180 or 900 minutes with lysis buffer in individual tubes. At the indicated times, one tube was removed, lysis buffer was replaced with ice-cold TE, and plugs were stored at 4°C until all plugs were ready for electrophoresis.  (F)  Quantification of kinetics of chromosomal fragmentation when plugs were made in the presence of RNase and lysed for 1, 5, 10, 30, 60, 180 or 900 minutes. Data points are means of three independent assays ± SEM. Arrow shows the value of fragmentation after 10 min lysis.
Figure Legend Snippet: Characterization of RiCF. (A) Representative radiogram showing RNase dose dependent chromosomal fragmentation in AB1157. Plugs were made with 0, 2, 10, 25, 50 or 100 μg RNase and lysed and electrophoresed under standard conditions. CZ, compression zone. (B) Quantification showing increase in chromosomal fragmentation in RNase dose-dependent manner. Data points are means of six independent assays ± SEM. (C) RNase-effect is not seen in the pre-lyzed cells. Plugs from AB1157 culture were made in the presence of proteinase K, but without any RNase. After overnight lysis and extensive washing, the plugs were incubated with 0, 2, 20 and 100 μg RNase or 100 U of EcoRI for 15 H at 37°C before PFGE. (D) Quantification of chromosomal fragmentation showing extreme sensitivity of chromosomes to EcoRI, but not RNase, when plugs were treated with the enzymes after lysis of cells. The experiment is done twice and a representative result is presented. (E) A representative radiogram showing kinetics of RiCF. Multiple plugs were made in the presence of RNase (50 μg/plug) and incubated at 62°C for 10, 30, 60, 180 or 900 minutes with lysis buffer in individual tubes. At the indicated times, one tube was removed, lysis buffer was replaced with ice-cold TE, and plugs were stored at 4°C until all plugs were ready for electrophoresis. (F) Quantification of kinetics of chromosomal fragmentation when plugs were made in the presence of RNase and lysed for 1, 5, 10, 30, 60, 180 or 900 minutes. Data points are means of three independent assays ± SEM. Arrow shows the value of fragmentation after 10 min lysis.

Techniques Used: Lysis, Incubation, Electrophoresis

Genetics of RiCF. (A)  Quantitative determination of RiCF in Δ hns  mutant. AB1157 and SRK254 were grown at 37°C to the same final OD, and plugs were made in the absence of proteinase K, but with or without RNase (50 μg/plug). After overnight incubation in the lysis buffer at 62°C, the plugs were electrophoresed under standard conditions. Data points are means of 6–10 independent assays± SEM.  (B)  Radiogram of a representative pulsed field gel from which data in (A) are calculated.  (C)  Comparison of RiCF in Δ hupA  Δ hupB  and Δ ihfA  Δ ihfB  double mutants. Experiment was done as described in (A), and values presented are means of 6–13 independent assays ± SEM.  (D)  Radiogram of a representative pulsed field gel from which data in (C) are calculated.  (E)  Effect of  hns  deletion on RiCF of Δ ihfA  Δ ihfB  mutant. Values presented are means of 7 independent assays ± SEM.
Figure Legend Snippet: Genetics of RiCF. (A) Quantitative determination of RiCF in Δ hns mutant. AB1157 and SRK254 were grown at 37°C to the same final OD, and plugs were made in the absence of proteinase K, but with or without RNase (50 μg/plug). After overnight incubation in the lysis buffer at 62°C, the plugs were electrophoresed under standard conditions. Data points are means of 6–10 independent assays± SEM. (B) Radiogram of a representative pulsed field gel from which data in (A) are calculated. (C) Comparison of RiCF in Δ hupA Δ hupB and Δ ihfA Δ ihfB double mutants. Experiment was done as described in (A), and values presented are means of 6–13 independent assays ± SEM. (D) Radiogram of a representative pulsed field gel from which data in (C) are calculated. (E) Effect of hns deletion on RiCF of Δ ihfA Δ ihfB mutant. Values presented are means of 7 independent assays ± SEM.

Techniques Used: Mutagenesis, Incubation, Lysis, Pulsed-Field Gel

Non-coding RNA and HU stabilize nucleoids. (A)  Comparison of spontaneous and RNase-induced fragmentation in Δ nc1  Δ nc5  Δ ihfA  Δ ihfB , Δ nc1  Δ nc5  Δ hupA  Δ hupB  and Δ nc1  Δ nc5  Δ hns  mutants. AB1157, SRK254-12, SRK254-15 and SRK254-18 were grown at 37°C, and plugs were made in absence of proteinase K, both with or without RNase (50 μg/plug), and lysed under standard conditions. Data points are means of at least three independent assays± SEM.  (B)  Radiogram of a representative gel from which data in (A) are generated.
Figure Legend Snippet: Non-coding RNA and HU stabilize nucleoids. (A) Comparison of spontaneous and RNase-induced fragmentation in Δ nc1 Δ nc5 Δ ihfA Δ ihfB , Δ nc1 Δ nc5 Δ hupA Δ hupB and Δ nc1 Δ nc5 Δ hns mutants. AB1157, SRK254-12, SRK254-15 and SRK254-18 were grown at 37°C, and plugs were made in absence of proteinase K, both with or without RNase (50 μg/plug), and lysed under standard conditions. Data points are means of at least three independent assays± SEM. (B) Radiogram of a representative gel from which data in (A) are generated.

Techniques Used: Generated

Endonuclease-I is critical for RiCF but not for spontaneous fragmentation. (A)  Comparison of spontaneous fragmentation and RiCF in AB1157, AB1157 Δ nc1  Δ nc5  Δ hupA  Δ hupB  and AB1157 Δ nc1  Δ nc5  Δ hupA  Δ hupB  Δ endA  mutants. All strains were grown at 37°C to the final OD of 0.6, and plugs were made in the absence of proteinase K, both with or without RNase (50 μg/plug). Data points are means of at least three independent assays ± SEM.  (B)  Radiogram of a representative gel from which data in (A) are generated.
Figure Legend Snippet: Endonuclease-I is critical for RiCF but not for spontaneous fragmentation. (A) Comparison of spontaneous fragmentation and RiCF in AB1157, AB1157 Δ nc1 Δ nc5 Δ hupA Δ hupB and AB1157 Δ nc1 Δ nc5 Δ hupA Δ hupB Δ endA mutants. All strains were grown at 37°C to the final OD of 0.6, and plugs were made in the absence of proteinase K, both with or without RNase (50 μg/plug). Data points are means of at least three independent assays ± SEM. (B) Radiogram of a representative gel from which data in (A) are generated.

Techniques Used: Generated

RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.
Figure Legend Snippet: RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

Techniques Used: Pulsed-Field Gel, Lysis

7) Product Images from "Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis"

Article Title: Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis

Journal: Nature Communications

doi: 10.1038/ncomms13932

Biochemical localization of the T. hominis ISC pathway components. ( a ) Western blots using antibodies to T. hominis proteins for fractions obtained by differential centrifugation from RK cells or RK cells infected with T. hominis (Th). Centrifugation speeds of pellet fractions are given above each lane. Sup=final 100,000 g (100 K) supernatant. ( b ) Western blot of the 25,000 g (25 K) pellet fraction of T. hominis -infected RK cells treated with or without proteinase K (PK) and Triton X-100 detergent as indicated.
Figure Legend Snippet: Biochemical localization of the T. hominis ISC pathway components. ( a ) Western blots using antibodies to T. hominis proteins for fractions obtained by differential centrifugation from RK cells or RK cells infected with T. hominis (Th). Centrifugation speeds of pellet fractions are given above each lane. Sup=final 100,000 g (100 K) supernatant. ( b ) Western blot of the 25,000 g (25 K) pellet fraction of T. hominis -infected RK cells treated with or without proteinase K (PK) and Triton X-100 detergent as indicated.

Techniques Used: Western Blot, Centrifugation, Infection

Localization of the T. hominis homologue of mitochondrial Atm1. ( a ) Fractionation by differential ultracentrifugation was carried out on healthy and T. hominis- infected RK cells and the fractions were immunostained using an antibody to ThAtm1_1. ( b ) Proteinase K (PK) protection was performed on the mitosome-enriched 25,000 g (25 K) fraction of infected cells, with Triton X-100 (TX) used to solubilize the membranes. A Coomassie-stained gel of the fractions after the protection assay is shown on the right. ( c – i ) Immunofluorescence of fixed RK-13 cells infected with T. hominis using antibodies raised against ThHsp70 ( c , e ; green; rat) or ThAtm1_1 ( d , e ; red; rabbit). DAPI was used to label host and parasite nuclear DNA (blue). The ThAtm1_1 antiserum was pre-purified using uninfected RK-13 cell lysate immobilized on nitrocellulose following SDS–PAGE. Colocalized pixels were identified using Zeiss Axiovision software, and are pseudo-coloured pink as shown in f . The DIC image in ( g ) shows the individual meronts in the infected RK cell. ( h , i ) A close-up of a different sample of meronts showing the typical distribution of the signals for the two antibodies. ( j ) Colocalisation scatter plots against the different channels were generated by Axiovision software and the antibody to the protein import receptor ThTom70 was used as a positive control for colocalisation with ThHsp70. The scatter plots for ThAtm1_1 with ThHsp70 and ThTom70 with ThHsp70 are similar, indicating co-localization of both proteins with ThHsp70. ( k – m ), The colocalized pixel counts were also quantified ( n =3, error bars=s.d.) and represented graphically for ThHsp70 and either ( k ) ThAtm1_1 or ( l ) ThTom70. ( m ) The extent of mitosomal labelling by the ThAtm1_1 antibody relative to other cell compartments was quantified based on the proportion of pixel intensity across six fields of view for each of three replicates (error bars=s.d.).
Figure Legend Snippet: Localization of the T. hominis homologue of mitochondrial Atm1. ( a ) Fractionation by differential ultracentrifugation was carried out on healthy and T. hominis- infected RK cells and the fractions were immunostained using an antibody to ThAtm1_1. ( b ) Proteinase K (PK) protection was performed on the mitosome-enriched 25,000 g (25 K) fraction of infected cells, with Triton X-100 (TX) used to solubilize the membranes. A Coomassie-stained gel of the fractions after the protection assay is shown on the right. ( c – i ) Immunofluorescence of fixed RK-13 cells infected with T. hominis using antibodies raised against ThHsp70 ( c , e ; green; rat) or ThAtm1_1 ( d , e ; red; rabbit). DAPI was used to label host and parasite nuclear DNA (blue). The ThAtm1_1 antiserum was pre-purified using uninfected RK-13 cell lysate immobilized on nitrocellulose following SDS–PAGE. Colocalized pixels were identified using Zeiss Axiovision software, and are pseudo-coloured pink as shown in f . The DIC image in ( g ) shows the individual meronts in the infected RK cell. ( h , i ) A close-up of a different sample of meronts showing the typical distribution of the signals for the two antibodies. ( j ) Colocalisation scatter plots against the different channels were generated by Axiovision software and the antibody to the protein import receptor ThTom70 was used as a positive control for colocalisation with ThHsp70. The scatter plots for ThAtm1_1 with ThHsp70 and ThTom70 with ThHsp70 are similar, indicating co-localization of both proteins with ThHsp70. ( k – m ), The colocalized pixel counts were also quantified ( n =3, error bars=s.d.) and represented graphically for ThHsp70 and either ( k ) ThAtm1_1 or ( l ) ThTom70. ( m ) The extent of mitosomal labelling by the ThAtm1_1 antibody relative to other cell compartments was quantified based on the proportion of pixel intensity across six fields of view for each of three replicates (error bars=s.d.).

Techniques Used: Fractionation, Infection, Staining, Immunofluorescence, Purification, SDS Page, Software, Generated, Positive Control

8) Product Images from "Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis"

Article Title: Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis

Journal: Nature Communications

doi: 10.1038/ncomms13932

Biochemical localization of the T. hominis ISC pathway components. ( a ) Western blots using antibodies to T. hominis proteins for fractions obtained by differential centrifugation from RK cells or RK cells infected with T. hominis (Th). Centrifugation speeds of pellet fractions are given above each lane. Sup=final 100,000 g (100 K) supernatant. ( b ) Western blot of the 25,000 g (25 K) pellet fraction of T. hominis -infected RK cells treated with or without proteinase K (PK) and Triton X-100 detergent as indicated.
Figure Legend Snippet: Biochemical localization of the T. hominis ISC pathway components. ( a ) Western blots using antibodies to T. hominis proteins for fractions obtained by differential centrifugation from RK cells or RK cells infected with T. hominis (Th). Centrifugation speeds of pellet fractions are given above each lane. Sup=final 100,000 g (100 K) supernatant. ( b ) Western blot of the 25,000 g (25 K) pellet fraction of T. hominis -infected RK cells treated with or without proteinase K (PK) and Triton X-100 detergent as indicated.

Techniques Used: Western Blot, Centrifugation, Infection

Localization of the T. hominis homologue of mitochondrial Atm1. ( a ) Fractionation by differential ultracentrifugation was carried out on healthy and T. hominis- infected RK cells and the fractions were immunostained using an antibody to ThAtm1_1. ( b ) Proteinase K (PK) protection was performed on the mitosome-enriched 25,000 g (25 K) fraction of infected cells, with Triton X-100 (TX) used to solubilize the membranes. A Coomassie-stained gel of the fractions after the protection assay is shown on the right. ( c – i ) Immunofluorescence of fixed RK-13 cells infected with T. hominis using antibodies raised against ThHsp70 ( c , e ; green; rat) or ThAtm1_1 ( d , e ; red; rabbit). DAPI was used to label host and parasite nuclear DNA (blue). The ThAtm1_1 antiserum was pre-purified using uninfected RK-13 cell lysate immobilized on nitrocellulose following SDS–PAGE. Colocalized pixels were identified using Zeiss Axiovision software, and are pseudo-coloured pink as shown in f . The DIC image in ( g ) shows the individual meronts in the infected RK cell. ( h , i ) A close-up of a different sample of meronts showing the typical distribution of the signals for the two antibodies. ( j ) Colocalisation scatter plots against the different channels were generated by Axiovision software and the antibody to the protein import receptor ThTom70 was used as a positive control for colocalisation with ThHsp70. The scatter plots for ThAtm1_1 with ThHsp70 and ThTom70 with ThHsp70 are similar, indicating co-localization of both proteins with ThHsp70. ( k – m ), The colocalized pixel counts were also quantified ( n =3, error bars=s.d.) and represented graphically for ThHsp70 and either ( k ) ThAtm1_1 or ( l ) ThTom70. ( m ) The extent of mitosomal labelling by the ThAtm1_1 antibody relative to other cell compartments was quantified based on the proportion of pixel intensity across six fields of view for each of three replicates (error bars=s.d.).
Figure Legend Snippet: Localization of the T. hominis homologue of mitochondrial Atm1. ( a ) Fractionation by differential ultracentrifugation was carried out on healthy and T. hominis- infected RK cells and the fractions were immunostained using an antibody to ThAtm1_1. ( b ) Proteinase K (PK) protection was performed on the mitosome-enriched 25,000 g (25 K) fraction of infected cells, with Triton X-100 (TX) used to solubilize the membranes. A Coomassie-stained gel of the fractions after the protection assay is shown on the right. ( c – i ) Immunofluorescence of fixed RK-13 cells infected with T. hominis using antibodies raised against ThHsp70 ( c , e ; green; rat) or ThAtm1_1 ( d , e ; red; rabbit). DAPI was used to label host and parasite nuclear DNA (blue). The ThAtm1_1 antiserum was pre-purified using uninfected RK-13 cell lysate immobilized on nitrocellulose following SDS–PAGE. Colocalized pixels were identified using Zeiss Axiovision software, and are pseudo-coloured pink as shown in f . The DIC image in ( g ) shows the individual meronts in the infected RK cell. ( h , i ) A close-up of a different sample of meronts showing the typical distribution of the signals for the two antibodies. ( j ) Colocalisation scatter plots against the different channels were generated by Axiovision software and the antibody to the protein import receptor ThTom70 was used as a positive control for colocalisation with ThHsp70. The scatter plots for ThAtm1_1 with ThHsp70 and ThTom70 with ThHsp70 are similar, indicating co-localization of both proteins with ThHsp70. ( k – m ), The colocalized pixel counts were also quantified ( n =3, error bars=s.d.) and represented graphically for ThHsp70 and either ( k ) ThAtm1_1 or ( l ) ThTom70. ( m ) The extent of mitosomal labelling by the ThAtm1_1 antibody relative to other cell compartments was quantified based on the proportion of pixel intensity across six fields of view for each of three replicates (error bars=s.d.).

Techniques Used: Fractionation, Infection, Staining, Immunofluorescence, Purification, SDS Page, Software, Generated, Positive Control

9) Product Images from "Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease"

Article Title: Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease

Journal: Brain

doi: 10.1093/brain/awq234

Immunoblot analysis of protease-resistant PrP TSE extracted from brains of capuchin monkeys (lanes 3–10) inoculated with different CJD subtypes or kuru. Brain homogenates were treated with proteinase K and probed with monoclonal antibody 9A2. Approximate
Figure Legend Snippet: Immunoblot analysis of protease-resistant PrP TSE extracted from brains of capuchin monkeys (lanes 3–10) inoculated with different CJD subtypes or kuru. Brain homogenates were treated with proteinase K and probed with monoclonal antibody 9A2. Approximate

Techniques Used:

( A ) Amount of proteinase K-resistant PrP TSE  in squirrel monkey total brain homogenates. The two groups of animals showing the PrP TSE  profile ‘a’ ( n =  56) or ‘b’ ( n =  16) on western blot are
Figure Legend Snippet: ( A ) Amount of proteinase K-resistant PrP TSE in squirrel monkey total brain homogenates. The two groups of animals showing the PrP TSE profile ‘a’ ( n =  56) or ‘b’ ( n =  16) on western blot are

Techniques Used: Western Blot

Immunoblot analysis of protease-resistant PrP TSE extracted from brains of squirrel monkeys (lanes 3–10) inoculated with different CJD subtypes or kuru. Brain homogenates were treated with proteinase K and probed with 3F4. Approximate molecular
Figure Legend Snippet: Immunoblot analysis of protease-resistant PrP TSE extracted from brains of squirrel monkeys (lanes 3–10) inoculated with different CJD subtypes or kuru. Brain homogenates were treated with proteinase K and probed with 3F4. Approximate molecular

Techniques Used:

10) Product Images from "Proteomic Characterization of Pseudorabies Virus Extracellular Virions ▿Proteomic Characterization of Pseudorabies Virus Extracellular Virions ▿ †"

Article Title: Proteomic Characterization of Pseudorabies Virus Extracellular Virions ▿Proteomic Characterization of Pseudorabies Virus Extracellular Virions ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.02253-10

Peptide sequence coverage of viral proteins in untreated and proteinase K-treated virions. Schematic representations of each protein are oriented with the N terminus on the left and the C terminus on the right. Regions of the viral proteins that correspond
Figure Legend Snippet: Peptide sequence coverage of viral proteins in untreated and proteinase K-treated virions. Schematic representations of each protein are oriented with the N terminus on the left and the C terminus on the right. Regions of the viral proteins that correspond

Techniques Used: Sequencing

Identification of viral and host virion proteins. (A) Mock, proteinase K-treated, or untreated purified virions prepared from 75-cm 2  PK15 cells that were PRV Becker infected or mock infected. Samples were run on a 4 to 12% 1D SDS-PAGE gel and stained
Figure Legend Snippet: Identification of viral and host virion proteins. (A) Mock, proteinase K-treated, or untreated purified virions prepared from 75-cm 2 PK15 cells that were PRV Becker infected or mock infected. Samples were run on a 4 to 12% 1D SDS-PAGE gel and stained

Techniques Used: Purification, Infection, SDS Page, Staining

Sensitivity of viral glycoproteins gC, gB, gI, and gB to proteinase K. Western blot analysis of viral proteins in cell lysates from mock-infected and PRV Becker-infected PK15 cells, and purified virions from mock, untreated, and proteinase K (ProK)-treated
Figure Legend Snippet: Sensitivity of viral glycoproteins gC, gB, gI, and gB to proteinase K. Western blot analysis of viral proteins in cell lysates from mock-infected and PRV Becker-infected PK15 cells, and purified virions from mock, untreated, and proteinase K (ProK)-treated

Techniques Used: Western Blot, Infection, Purification

Functional classification of host proteins in PRV virions. We identified a total of 48 host proteins that were present in untreated and proteinase K-treated virions but not in samples from mock-infected cells. These proteins were manually classified according
Figure Legend Snippet: Functional classification of host proteins in PRV virions. We identified a total of 48 host proteins that were present in untreated and proteinase K-treated virions but not in samples from mock-infected cells. These proteins were manually classified according

Techniques Used: Functional Assay, Infection

11) Product Images from "Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing"

Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing

Journal: Scientific Reports

doi: 10.1038/srep24228

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with a mono-nucleosome. ( a ) Scheme of the ternary complex formation assay. ( b, c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with ssDNA-conjugated magnetic beads (final 5 μM in nucleotides). A heterologous poly dT 80-mer or a homologous ssDNA 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–8) or mono-nucleosomes (lanes 9–16) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, 9, and 13 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with a mono-nucleosome. ( a ) Scheme of the ternary complex formation assay. ( b, c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with ssDNA-conjugated magnetic beads (final 5 μM in nucleotides). A heterologous poly dT 80-mer or a homologous ssDNA 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–8) or mono-nucleosomes (lanes 9–16) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, 9, and 13 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.

Techniques Used: Tube Formation Assay, Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining

Competitive homologous-pairing assay. ( a ) Scheme of the competitive homologous-pairing assay. Asterisks indicate the  32 P-labeled 5′-end of the 5 S  ssDNA 70-mer. ( b ) The D-loop formation assay on the 5 S  DNA sequences. RAD51 (0.4 μM) or DMC1 (0.4 μM) was incubated with the 5 S  ssDNA 70-mer (final 1 μM in nucleotides). The reactions were conducted in the presence of HOP2-MND1 (denoted as H2M1). The reaction was initiated by adding the naked dsDNA (final 30 μM in nucleotides) in the presence of the competitor nucleosome. The nucleosome concentrations are indicated at the top of the panel. After a 10 min incubation, the reaction was stopped by SDS and proteinase K, and the reaction products were separated by agarose gel electrophoresis. ( c ) Graphic representation of the experiments shown in panel ( b ). The amounts of D-loop formation relative to RAD51 or DMC1 with HOP2-MND1 (lanes 2 and 7 of panel ( b )) are plotted against the nucleosome concentrations. The average values of four independent experiments are shown with the SD values.
Figure Legend Snippet: Competitive homologous-pairing assay. ( a ) Scheme of the competitive homologous-pairing assay. Asterisks indicate the 32 P-labeled 5′-end of the 5 S ssDNA 70-mer. ( b ) The D-loop formation assay on the 5 S DNA sequences. RAD51 (0.4 μM) or DMC1 (0.4 μM) was incubated with the 5 S ssDNA 70-mer (final 1 μM in nucleotides). The reactions were conducted in the presence of HOP2-MND1 (denoted as H2M1). The reaction was initiated by adding the naked dsDNA (final 30 μM in nucleotides) in the presence of the competitor nucleosome. The nucleosome concentrations are indicated at the top of the panel. After a 10 min incubation, the reaction was stopped by SDS and proteinase K, and the reaction products were separated by agarose gel electrophoresis. ( c ) Graphic representation of the experiments shown in panel ( b ). The amounts of D-loop formation relative to RAD51 or DMC1 with HOP2-MND1 (lanes 2 and 7 of panel ( b )) are plotted against the nucleosome concentrations. The average values of four independent experiments are shown with the SD values.

Techniques Used: Labeling, Tube Formation Assay, Incubation, Agarose Gel Electrophoresis

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with the nucleosome lacking the N-terminal histone tails. ( a ) RAD51 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). A heterologous poly dT 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–4), wild-type mono-nucleosomes (lanes 5–8), or all tailless mono-nucleosomes (lanes 9–12) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, and 9 were performed in the absence of RAD51 and DMC1. ( b ) Graphic representation of the experiments shown in panel ( a ). The amounts of the ternary complex formation are plotted against the RAD51 concentration. The average values of three independent experiments are shown with the SD values. ( c ) DMC1 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). The experiments were performed by the same procedure as in panel ( a ). ( d ) Graphic representation of the experiments shown in panel ( c ). The amounts of the ternary complex formation are plotted against the DMC1 concentration. The average values of three independent experiments are shown with the SD values.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with the nucleosome lacking the N-terminal histone tails. ( a ) RAD51 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). A heterologous poly dT 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–4), wild-type mono-nucleosomes (lanes 5–8), or all tailless mono-nucleosomes (lanes 9–12) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, and 9 were performed in the absence of RAD51 and DMC1. ( b ) Graphic representation of the experiments shown in panel ( a ). The amounts of the ternary complex formation are plotted against the RAD51 concentration. The average values of three independent experiments are shown with the SD values. ( c ) DMC1 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). The experiments were performed by the same procedure as in panel ( a ). ( d ) Graphic representation of the experiments shown in panel ( c ). The amounts of the ternary complex formation are plotted against the DMC1 concentration. The average values of three independent experiments are shown with the SD values.

Techniques Used: Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining, Concentration Assay

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with nucleosome arrays. ( a ) Scheme of the ternary complex formation assay with the nucleosome arrays. ( b,c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with the ssDNA (poly dT 80-mer)-conjugated magnetic beads in the presence of HOP2-MND1 (denoted as H2M1). After a 5 min incubation, naked dsDNA (lanes 1–5), tri-nucleosomes (lanes 6–10), or di-nucleosomes (lanes 11–15) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 6, and 11 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with nucleosome arrays. ( a ) Scheme of the ternary complex formation assay with the nucleosome arrays. ( b,c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with the ssDNA (poly dT 80-mer)-conjugated magnetic beads in the presence of HOP2-MND1 (denoted as H2M1). After a 5 min incubation, naked dsDNA (lanes 1–5), tri-nucleosomes (lanes 6–10), or di-nucleosomes (lanes 11–15) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 6, and 11 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.

Techniques Used: Tube Formation Assay, Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining

12) Product Images from "Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing"

Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing

Journal: Scientific Reports

doi: 10.1038/srep24228

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with a mono-nucleosome. ( a ) Scheme of the ternary complex formation assay. ( b, c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with ssDNA-conjugated magnetic beads (final 5 μM in nucleotides). A heterologous poly dT 80-mer or a homologous ssDNA 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–8) or mono-nucleosomes (lanes 9–16) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, 9, and 13 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with a mono-nucleosome. ( a ) Scheme of the ternary complex formation assay. ( b, c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with ssDNA-conjugated magnetic beads (final 5 μM in nucleotides). A heterologous poly dT 80-mer or a homologous ssDNA 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–8) or mono-nucleosomes (lanes 9–16) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, 9, and 13 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.

Techniques Used: Tube Formation Assay, Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining

Competitive homologous-pairing assay. ( a ) Scheme of the competitive homologous-pairing assay. Asterisks indicate the  32 P-labeled 5′-end of the 5 S  ssDNA 70-mer. ( b ) The D-loop formation assay on the 5 S  DNA sequences. RAD51 (0.4 μM) or DMC1 (0.4 μM) was incubated with the 5 S  ssDNA 70-mer (final 1 μM in nucleotides). The reactions were conducted in the presence of HOP2-MND1 (denoted as H2M1). The reaction was initiated by adding the naked dsDNA (final 30 μM in nucleotides) in the presence of the competitor nucleosome. The nucleosome concentrations are indicated at the top of the panel. After a 10 min incubation, the reaction was stopped by SDS and proteinase K, and the reaction products were separated by agarose gel electrophoresis. ( c ) Graphic representation of the experiments shown in panel ( b ). The amounts of D-loop formation relative to RAD51 or DMC1 with HOP2-MND1 (lanes 2 and 7 of panel ( b )) are plotted against the nucleosome concentrations. The average values of four independent experiments are shown with the SD values.
Figure Legend Snippet: Competitive homologous-pairing assay. ( a ) Scheme of the competitive homologous-pairing assay. Asterisks indicate the 32 P-labeled 5′-end of the 5 S ssDNA 70-mer. ( b ) The D-loop formation assay on the 5 S DNA sequences. RAD51 (0.4 μM) or DMC1 (0.4 μM) was incubated with the 5 S ssDNA 70-mer (final 1 μM in nucleotides). The reactions were conducted in the presence of HOP2-MND1 (denoted as H2M1). The reaction was initiated by adding the naked dsDNA (final 30 μM in nucleotides) in the presence of the competitor nucleosome. The nucleosome concentrations are indicated at the top of the panel. After a 10 min incubation, the reaction was stopped by SDS and proteinase K, and the reaction products were separated by agarose gel electrophoresis. ( c ) Graphic representation of the experiments shown in panel ( b ). The amounts of D-loop formation relative to RAD51 or DMC1 with HOP2-MND1 (lanes 2 and 7 of panel ( b )) are plotted against the nucleosome concentrations. The average values of four independent experiments are shown with the SD values.

Techniques Used: Labeling, Tube Formation Assay, Incubation, Agarose Gel Electrophoresis

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with the nucleosome lacking the N-terminal histone tails. ( a ) RAD51 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). A heterologous poly dT 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–4), wild-type mono-nucleosomes (lanes 5–8), or all tailless mono-nucleosomes (lanes 9–12) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, and 9 were performed in the absence of RAD51 and DMC1. ( b ) Graphic representation of the experiments shown in panel ( a ). The amounts of the ternary complex formation are plotted against the RAD51 concentration. The average values of three independent experiments are shown with the SD values. ( c ) DMC1 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). The experiments were performed by the same procedure as in panel ( a ). ( d ) Graphic representation of the experiments shown in panel ( c ). The amounts of the ternary complex formation are plotted against the DMC1 concentration. The average values of three independent experiments are shown with the SD values.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with the nucleosome lacking the N-terminal histone tails. ( a ) RAD51 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). A heterologous poly dT 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–4), wild-type mono-nucleosomes (lanes 5–8), or all tailless mono-nucleosomes (lanes 9–12) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, and 9 were performed in the absence of RAD51 and DMC1. ( b ) Graphic representation of the experiments shown in panel ( a ). The amounts of the ternary complex formation are plotted against the RAD51 concentration. The average values of three independent experiments are shown with the SD values. ( c ) DMC1 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). The experiments were performed by the same procedure as in panel ( a ). ( d ) Graphic representation of the experiments shown in panel ( c ). The amounts of the ternary complex formation are plotted against the DMC1 concentration. The average values of three independent experiments are shown with the SD values.

Techniques Used: Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining, Concentration Assay

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with nucleosome arrays. ( a ) Scheme of the ternary complex formation assay with the nucleosome arrays. ( b,c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with the ssDNA (poly dT 80-mer)-conjugated magnetic beads in the presence of HOP2-MND1 (denoted as H2M1). After a 5 min incubation, naked dsDNA (lanes 1–5), tri-nucleosomes (lanes 6–10), or di-nucleosomes (lanes 11–15) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 6, and 11 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with nucleosome arrays. ( a ) Scheme of the ternary complex formation assay with the nucleosome arrays. ( b,c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with the ssDNA (poly dT 80-mer)-conjugated magnetic beads in the presence of HOP2-MND1 (denoted as H2M1). After a 5 min incubation, naked dsDNA (lanes 1–5), tri-nucleosomes (lanes 6–10), or di-nucleosomes (lanes 11–15) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 6, and 11 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.

Techniques Used: Tube Formation Assay, Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining

13) Product Images from "Scrapie-like prion protein accumulates in aggresomes of cyclosporin A-treated cells"

Article Title: Scrapie-like prion protein accumulates in aggresomes of cyclosporin A-treated cells

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg045

Fig. 1.  Protease-resistant and detergent-insoluble PrP species accumulate in CsA-treated cells (western blots). N2a-M cells were incubated with CsA as detailed below, and then lysed either in Triton-doc (A, C and D) or NOG (B) lysis buffers. Post nuclear supernatants (PNS) were then subjected to proteolysis or sedimentation as detailed below, and the PrP species were analyzed in western blots developed with the PrP mAb 3F4. ( A ) Cells were incubated with CsA at the indicated concentration for 24 h, and the lysates were analyzed with (+PK) or without (–PK) prior stringent proteolysis (20 µg/ml proteinase K, 30 min, 37°C). A major 26 kDa PrP species accumulated at high CsA concentrations (–PK, left panel, arrow). This band probably represents full-length, unglycosylated PrP. Proteolysis generated a major protease-resistant core of 19 kDa (+PK, right panel, arrowhead). ( B ) Cells were incubated for 36 h with or without 20 µg/ml CsA, as indicated. PNS were made 1% with Sarkosyl and centrifuged through 10–60% sucrose gradients containing 1% Sarkosyl. The CsA-induced 26 kDa species sedimented to the bottom of the gradient (arrow). ( C ) Cells were incubated with 15 µg/ml CsA for the indicated time. PNS were made 1% with Sarkosyl and separated into high-speed supernatants (not shown) and pellets. Densitometry of the 26 kDa band shows first a slight decrease and then a significant increase in the sedimenting PrP species (four independent experiments). ( D ) Cells were incubated for 6 h with the indicated CsA concentrations, and high-speed pellets were prepared as in (C). The insoluble PrP species increased until the CsA concentration reached 60 µg/ml, and then leveled off.
Figure Legend Snippet: Fig. 1. Protease-resistant and detergent-insoluble PrP species accumulate in CsA-treated cells (western blots). N2a-M cells were incubated with CsA as detailed below, and then lysed either in Triton-doc (A, C and D) or NOG (B) lysis buffers. Post nuclear supernatants (PNS) were then subjected to proteolysis or sedimentation as detailed below, and the PrP species were analyzed in western blots developed with the PrP mAb 3F4. ( A ) Cells were incubated with CsA at the indicated concentration for 24 h, and the lysates were analyzed with (+PK) or without (–PK) prior stringent proteolysis (20 µg/ml proteinase K, 30 min, 37°C). A major 26 kDa PrP species accumulated at high CsA concentrations (–PK, left panel, arrow). This band probably represents full-length, unglycosylated PrP. Proteolysis generated a major protease-resistant core of 19 kDa (+PK, right panel, arrowhead). ( B ) Cells were incubated for 36 h with or without 20 µg/ml CsA, as indicated. PNS were made 1% with Sarkosyl and centrifuged through 10–60% sucrose gradients containing 1% Sarkosyl. The CsA-induced 26 kDa species sedimented to the bottom of the gradient (arrow). ( C ) Cells were incubated with 15 µg/ml CsA for the indicated time. PNS were made 1% with Sarkosyl and separated into high-speed supernatants (not shown) and pellets. Densitometry of the 26 kDa band shows first a slight decrease and then a significant increase in the sedimenting PrP species (four independent experiments). ( D ) Cells were incubated for 6 h with the indicated CsA concentrations, and high-speed pellets were prepared as in (C). The insoluble PrP species increased until the CsA concentration reached 60 µg/ml, and then leveled off.

Techniques Used: Western Blot, Incubation, Lysis, Sedimentation, Concentration Assay, Generated

Fig. 2.  CsA-induced PrP: kinetics of accumulation, proteasome resistance and comparison with PrP Sc . (A–C) N2a-M cells were incubated with 150 µM ALLN, 25 µg/ml CsA, or both inhibitors (as indicated), for the indicated time. Sarkosyl-insoluble PrP species were then analyzed in western blots, which were first developed with 3F4 (A) and then reprobed with a ubiquitin (ub) mAb (C). Densitometry of the 26 kDa in (A) is shown in (B) (four independent experiments). ( A  and  B ) In contrast to CsA, ALLN caused an immediate increase in insoluble 26 kDa PrP. ( C ) ALLN, but not CsA, induced the accumulation of insoluble poly-ub conjugates (presumably proteasome bound). Since CsA did not inhibit the ubiquitylation machinery (ALLN + CsA), the lack of poly-ub conjugates with CsA alone indicates that proteasomes were not inhibited by this drug. ( D ) N2a-M cells were treated for 24 h with ALLN (75 µM), CsA (25 µg/ml) or left untreated (cont). PNS were separated into high-speed pellets (upper panel) and supernatants (data not shown) or subjected to stringent proteolysis (middle and bottom panels). The ALLN- and CsA-induced 26 and 22 kDa PrP bands co-migrated with the equivalent species in untreated cells. The 19 kDa protease-resistant cores elicited by CsA and ALLN also co-migrated, both when probed with 3F4 or with the C-terminal PrP antiserum R009. ( E ) N2a-M cells were either treated for 24 h with 25 µg/ml CsA or left untreated. Sarkosyl-insoluble fractions (lanes 3 and 6) were prepared by ultracentrifugation as described above. Lanes 1, 2, 4 and 5: total cell membranes were prepared from parallel cultures and extracted with sodium carbonate. Three-quarters of each membrane preparation was enzymatically deglycosylated with PNGase F (lanes 2 and 5), while the remaining membranes were analyzed without further treatment (lanes 1 and 4). That the Sarkosyl-insoluble 26 kDa bands strictly co-migrate with the deglycosylated membranal PrP confirms the ER origin of the former PrP species. ( F ) Comparison with PrP Sc . The proteinase K-resistant core (20 µg/ml proteinase K, 30 min, 37°C) of CsA-treated (25 µg/ml CsA, 24 h) N2a-M cells (lanes 1 and 3) is smaller than that of the prion isoform PrP Sc  in untreated ScN2a-M cells (lanes 2 and 5). In ScN2a-M cells treated with CsA (lane 4), these two PK-resistant cores appeared as a doublet. ( G ) Histogram: denaturation-dependent immunoassay. Untreated, ALLN- (75 µM, 24 h) and CsA- (25 µg/ml, 24 h) treated N2a-M cells, untreated ScN2a-M cells and an uninfected hamster brain were lysed and spotted on nitrocellulose strips that were incubated with the indicated concentration of GdnSCN prior to development with 3F4. The ALLN and CsA samples, as well as the ScN2a-M lysates, were subjected to proteolysis (20 µg/ml, 37°C, 30 min) prior to blotting. The ALLN and CsA samples resembled the scrapie samples in that denaturation increased their immunoreactivity, in contrast to the PrP C  present in the untreated N2a-M and the control brain.
Figure Legend Snippet: Fig. 2. CsA-induced PrP: kinetics of accumulation, proteasome resistance and comparison with PrP Sc . (A–C) N2a-M cells were incubated with 150 µM ALLN, 25 µg/ml CsA, or both inhibitors (as indicated), for the indicated time. Sarkosyl-insoluble PrP species were then analyzed in western blots, which were first developed with 3F4 (A) and then reprobed with a ubiquitin (ub) mAb (C). Densitometry of the 26 kDa in (A) is shown in (B) (four independent experiments). ( A and B ) In contrast to CsA, ALLN caused an immediate increase in insoluble 26 kDa PrP. ( C ) ALLN, but not CsA, induced the accumulation of insoluble poly-ub conjugates (presumably proteasome bound). Since CsA did not inhibit the ubiquitylation machinery (ALLN + CsA), the lack of poly-ub conjugates with CsA alone indicates that proteasomes were not inhibited by this drug. ( D ) N2a-M cells were treated for 24 h with ALLN (75 µM), CsA (25 µg/ml) or left untreated (cont). PNS were separated into high-speed pellets (upper panel) and supernatants (data not shown) or subjected to stringent proteolysis (middle and bottom panels). The ALLN- and CsA-induced 26 and 22 kDa PrP bands co-migrated with the equivalent species in untreated cells. The 19 kDa protease-resistant cores elicited by CsA and ALLN also co-migrated, both when probed with 3F4 or with the C-terminal PrP antiserum R009. ( E ) N2a-M cells were either treated for 24 h with 25 µg/ml CsA or left untreated. Sarkosyl-insoluble fractions (lanes 3 and 6) were prepared by ultracentrifugation as described above. Lanes 1, 2, 4 and 5: total cell membranes were prepared from parallel cultures and extracted with sodium carbonate. Three-quarters of each membrane preparation was enzymatically deglycosylated with PNGase F (lanes 2 and 5), while the remaining membranes were analyzed without further treatment (lanes 1 and 4). That the Sarkosyl-insoluble 26 kDa bands strictly co-migrate with the deglycosylated membranal PrP confirms the ER origin of the former PrP species. ( F ) Comparison with PrP Sc . The proteinase K-resistant core (20 µg/ml proteinase K, 30 min, 37°C) of CsA-treated (25 µg/ml CsA, 24 h) N2a-M cells (lanes 1 and 3) is smaller than that of the prion isoform PrP Sc in untreated ScN2a-M cells (lanes 2 and 5). In ScN2a-M cells treated with CsA (lane 4), these two PK-resistant cores appeared as a doublet. ( G ) Histogram: denaturation-dependent immunoassay. Untreated, ALLN- (75 µM, 24 h) and CsA- (25 µg/ml, 24 h) treated N2a-M cells, untreated ScN2a-M cells and an uninfected hamster brain were lysed and spotted on nitrocellulose strips that were incubated with the indicated concentration of GdnSCN prior to development with 3F4. The ALLN and CsA samples, as well as the ScN2a-M lysates, were subjected to proteolysis (20 µg/ml, 37°C, 30 min) prior to blotting. The ALLN and CsA samples resembled the scrapie samples in that denaturation increased their immunoreactivity, in contrast to the PrP C present in the untreated N2a-M and the control brain.

Techniques Used: Incubation, Western Blot, Concentration Assay

Fig. 4. The PrP aggresomes co-localize with γ-tubulin and 20S proteasomes but not with ubiquitin; they are inhibited by nocodazole and contain protease-resistant PrP. ( A and B ) CHO-M cells were incubated for 24 h either with or without 30 µg/ml CsA, as indicated, and examined by immunofluorescent confocal microscopy. PrP was detected using either RO73 (A, green channel) or 3F4 (B, red) and additional Abs as indicated. PrP aggresomes co-localized with the centrosomal marker γ-tubulin (A) and with 20S proteasomes (B) but not with ub (A), and they were found in the general region of the cells that stained with wheat germ agglutinin (WGA) (B). ( C ) CHO-M cells were incubated for 24 h either with 10 µg/ml nocodazole, with 30 µg/ml CsA and 10 µg/ml nocodazole or with 30 µg/ml CsA and 75 µM ALLN, and then examined by immunofluorescence for PrP (R073, green) and vimentin (red). Nocodazole prevented the formation of PrP aggresomes by CsA, but multiple PrP aggregates could be seen throughout the cell and especially underneath the cell surface (arrowheads). ALLN and CsA induced diffuse cytosolic accumulation as well as a single juxtanuclear, vimentin-caged deposit of PrP (arrowhead). ( D ) N2a-M cells treated for 24 h either with 25 µg/ml CsA, or with 25 µg/ml CsA and 10 µg/ml nocodazole, were subjected to proteolysis (20 µg/ml proteinase K, 30 min, 37°C) prior to western blotting with 3F4. Nocodazole did not prevent the formation of PK-resistant PrP, and the formation of aggresomes is thus not a prerequisite for the protease resistance of the CsA-induced PrP species. ( E ) CHO-M cells were treated with CsA (30 µM, 48 h), and then fixed with formalin and incubated with proteinase K (right panel; 7.5 µg/ml, 30 min, 37°C) prior to immunodetection of PrP with RO73 (red). The PrP immunoreactivity in aggresomes was protease resistant.
Figure Legend Snippet: Fig. 4. The PrP aggresomes co-localize with γ-tubulin and 20S proteasomes but not with ubiquitin; they are inhibited by nocodazole and contain protease-resistant PrP. ( A and B ) CHO-M cells were incubated for 24 h either with or without 30 µg/ml CsA, as indicated, and examined by immunofluorescent confocal microscopy. PrP was detected using either RO73 (A, green channel) or 3F4 (B, red) and additional Abs as indicated. PrP aggresomes co-localized with the centrosomal marker γ-tubulin (A) and with 20S proteasomes (B) but not with ub (A), and they were found in the general region of the cells that stained with wheat germ agglutinin (WGA) (B). ( C ) CHO-M cells were incubated for 24 h either with 10 µg/ml nocodazole, with 30 µg/ml CsA and 10 µg/ml nocodazole or with 30 µg/ml CsA and 75 µM ALLN, and then examined by immunofluorescence for PrP (R073, green) and vimentin (red). Nocodazole prevented the formation of PrP aggresomes by CsA, but multiple PrP aggregates could be seen throughout the cell and especially underneath the cell surface (arrowheads). ALLN and CsA induced diffuse cytosolic accumulation as well as a single juxtanuclear, vimentin-caged deposit of PrP (arrowhead). ( D ) N2a-M cells treated for 24 h either with 25 µg/ml CsA, or with 25 µg/ml CsA and 10 µg/ml nocodazole, were subjected to proteolysis (20 µg/ml proteinase K, 30 min, 37°C) prior to western blotting with 3F4. Nocodazole did not prevent the formation of PK-resistant PrP, and the formation of aggresomes is thus not a prerequisite for the protease resistance of the CsA-induced PrP species. ( E ) CHO-M cells were treated with CsA (30 µM, 48 h), and then fixed with formalin and incubated with proteinase K (right panel; 7.5 µg/ml, 30 min, 37°C) prior to immunodetection of PrP with RO73 (red). The PrP immunoreactivity in aggresomes was protease resistant.

Techniques Used: Incubation, Confocal Microscopy, Marker, Staining, Whole Genome Amplification, Immunofluorescence, Western Blot, Immunodetection

14) Product Images from "The Primary Folding Defect and Rescue of ?F508 CFTR Emerge during Translation of the Mutant Domain"

Article Title: The Primary Folding Defect and Rescue of ?F508 CFTR Emerge during Translation of the Mutant Domain

Journal: PLoS ONE

doi: 10.1371/journal.pone.0015458

Rescue of NBD1 conformation by the I539T suppressor mutation. (A) Wild-type and ΔF508 NBD1 (top panel) mRNAs containing the G550E (middle panel) or I539T (bottom panel) mutation were in vitro translated in the presence of 35 S-labeled methionine and cysteine and analyzed by 15% SDS-PAGE after proteinase K treatment. Asterisk indicates the 27 kDa fragment, arrowhead indicates the 25 kDa fragment. (B) Longer exposure of the 100 µg/ml proteinase K digest of in vitro translated NBD1, from same experiment as shown in B, showing the rescue of the 17 kDa band by the I539T but not by the G550E mutation. Gel lanes are aligned on the 25 kDa bands. (C) CFTR molecules containing the indicated mutations were in vitro translated, analyzed using 12% SDS-PAGE and lanes were quantified as described in Figure 1B . The arrowhead indicates the 25 kDa fragment, which has slightly decreased mobility when the I539T mutation is present.
Figure Legend Snippet: Rescue of NBD1 conformation by the I539T suppressor mutation. (A) Wild-type and ΔF508 NBD1 (top panel) mRNAs containing the G550E (middle panel) or I539T (bottom panel) mutation were in vitro translated in the presence of 35 S-labeled methionine and cysteine and analyzed by 15% SDS-PAGE after proteinase K treatment. Asterisk indicates the 27 kDa fragment, arrowhead indicates the 25 kDa fragment. (B) Longer exposure of the 100 µg/ml proteinase K digest of in vitro translated NBD1, from same experiment as shown in B, showing the rescue of the 17 kDa band by the I539T but not by the G550E mutation. Gel lanes are aligned on the 25 kDa bands. (C) CFTR molecules containing the indicated mutations were in vitro translated, analyzed using 12% SDS-PAGE and lanes were quantified as described in Figure 1B . The arrowhead indicates the 25 kDa fragment, which has slightly decreased mobility when the I539T mutation is present.

Techniques Used: Mutagenesis, In Vitro, Labeling, SDS Page

Minimal and local misfolding of ΔF508 CFTR. (A) Both CFTR and ΔF508 CFTR were translated in vitro in the presence of 35 S-methionine and cysteine and semi-permeabilized HT1080 cells for 60 min. Cells containing radiolabeled CFTR proteins were washed, lysed in Triton X-100, and prepared for limited proteolysis using increasing concentrations of proteinase K. The proteolytic digests were analyzed by 12% SDS-PAGE. The conformational difference between wild-type CFTR and ΔF508 CFTR is indicated by an arrowhead. (B) Relative intensities of all protease resistant fragments from a total 5 µg/ml Proteinase K digest, as in Figure 1A, were determined by total lane quantitation (Quantity One software Biorad). The y-axis represents electrophoretic mobility in 12% SDS-PA gel and the x-axis the relative intensity of the protease resistant fragments. The horizontal lines indicate the structural differences as described in A. The horizontal line indicated with an asterisk represents yet unidentified changes in the proteolytic pattern as a result of the ΔF508 mutation. The bracket represents small proteolytic fractions detected in both mutants. (C) Wild-type and ΔF508 CFTR were synthesized as in a, were subjected to 5 µg/ml proteinase K and NBD1-originated fragments were immunoprecipitated with polyclonal antibodies directed against NBD1 (Mr Pink) or against the R-region (G449). Arrowhead marks the NBD1-related fragment.
Figure Legend Snippet: Minimal and local misfolding of ΔF508 CFTR. (A) Both CFTR and ΔF508 CFTR were translated in vitro in the presence of 35 S-methionine and cysteine and semi-permeabilized HT1080 cells for 60 min. Cells containing radiolabeled CFTR proteins were washed, lysed in Triton X-100, and prepared for limited proteolysis using increasing concentrations of proteinase K. The proteolytic digests were analyzed by 12% SDS-PAGE. The conformational difference between wild-type CFTR and ΔF508 CFTR is indicated by an arrowhead. (B) Relative intensities of all protease resistant fragments from a total 5 µg/ml Proteinase K digest, as in Figure 1A, were determined by total lane quantitation (Quantity One software Biorad). The y-axis represents electrophoretic mobility in 12% SDS-PA gel and the x-axis the relative intensity of the protease resistant fragments. The horizontal lines indicate the structural differences as described in A. The horizontal line indicated with an asterisk represents yet unidentified changes in the proteolytic pattern as a result of the ΔF508 mutation. The bracket represents small proteolytic fractions detected in both mutants. (C) Wild-type and ΔF508 CFTR were synthesized as in a, were subjected to 5 µg/ml proteinase K and NBD1-originated fragments were immunoprecipitated with polyclonal antibodies directed against NBD1 (Mr Pink) or against the R-region (G449). Arrowhead marks the NBD1-related fragment.

Techniques Used: In Vitro, SDS Page, Quantitation Assay, Software, Mutagenesis, Synthesized, Immunoprecipitation

The effect of ΔF508 mutation on NBD1 alone. (A) Wild-type and ΔF508 NBD1 were in vitro translated for 30 min, treated with indicated proteinase K concentrations as in Figure 1 , and analyzed using 15% SDS-PAGE. The full length NBD1 domain is indicated by “#”, the asterisk (*) indicates the 27 kDa fragment, arrowhead (◂) indicates the 25 kDa fragment. (B) Similar experimental conditions as described in A, but using TPCK-trypsin as protease. A bracket (]) marks the triplet of protease resistant fragments and the dot (•) marks the 17 kDa fragment. (C) Wild-type and ΔF508 NBD1 were synthesized as in A, treated with 100 µg/ml trypsin, and fragments were immunoprecipitated with antibody 7D12 against NBD1. Fragments are labeled similar as in B. (D) Wild-type NBD1 was synthesized as in A, treated with 25 µg/ml proteinase K, and fragments were immunoprecipitated with the 7D12, 3G11 and Mr Pink antibody, recognizing specific epitopes within NBD1. Fragments are labeled similar as in A. (E) CHO cells expressing wild-type or ΔF508 NBD1 were pulse-labeled with 35 S-methionine and cysteine for 5 min and chased for indicated times. NBD1 was immunoprecipitated using polyclonal antibody Mr Pink and analyzed using 15% SDS-PAGE. NBD1 indicated by “#”. (F) Purified human wild-type and ΔF508 NBD1, indicated by “#”, were incubated with 2 µg/ml Proteinase K for 0, 2, 5, 10 and 30 minutes at room temperature. Proteolytic digests were separated using 15% SDS-PAGE and visualized by silver staining. Asterisk (*) and arrowhead (◂) indicate 27 and 25 kDa fragments resp., and are similar as in A. The open arrowhead (
Figure Legend Snippet: The effect of ΔF508 mutation on NBD1 alone. (A) Wild-type and ΔF508 NBD1 were in vitro translated for 30 min, treated with indicated proteinase K concentrations as in Figure 1 , and analyzed using 15% SDS-PAGE. The full length NBD1 domain is indicated by “#”, the asterisk (*) indicates the 27 kDa fragment, arrowhead (◂) indicates the 25 kDa fragment. (B) Similar experimental conditions as described in A, but using TPCK-trypsin as protease. A bracket (]) marks the triplet of protease resistant fragments and the dot (•) marks the 17 kDa fragment. (C) Wild-type and ΔF508 NBD1 were synthesized as in A, treated with 100 µg/ml trypsin, and fragments were immunoprecipitated with antibody 7D12 against NBD1. Fragments are labeled similar as in B. (D) Wild-type NBD1 was synthesized as in A, treated with 25 µg/ml proteinase K, and fragments were immunoprecipitated with the 7D12, 3G11 and Mr Pink antibody, recognizing specific epitopes within NBD1. Fragments are labeled similar as in A. (E) CHO cells expressing wild-type or ΔF508 NBD1 were pulse-labeled with 35 S-methionine and cysteine for 5 min and chased for indicated times. NBD1 was immunoprecipitated using polyclonal antibody Mr Pink and analyzed using 15% SDS-PAGE. NBD1 indicated by “#”. (F) Purified human wild-type and ΔF508 NBD1, indicated by “#”, were incubated with 2 µg/ml Proteinase K for 0, 2, 5, 10 and 30 minutes at room temperature. Proteolytic digests were separated using 15% SDS-PAGE and visualized by silver staining. Asterisk (*) and arrowhead (◂) indicate 27 and 25 kDa fragments resp., and are similar as in A. The open arrowhead (

Techniques Used: Mutagenesis, In Vitro, SDS Page, Synthesized, Immunoprecipitation, Labeling, Expressing, Purification, Incubation, Silver Staining

15) Product Images from "Structural and functional characterization of two alpha-synuclein strains"

Article Title: Structural and functional characterization of two alpha-synuclein strains

Journal: Nature Communications

doi: 10.1038/ncomms3575

Cross-seeding capacities of the two α-syn polymorphs. Elongation of preformed α-syn fibrils (red data points) and ribbons (blue data points), 10 μM in ( a ) buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl), ( b ) buffer B (5 mM Tris-HCl, pH 7.5), at 37 °C, in the presence of soluble α-syn (100 μM), monitored by measurement of scattered light at 440 nm. The control assembly reactions of soluble α-syn in the absence of preformed seeds (black data points) are also shown. When the seeded assembly reactions were centrifuged immediately after addition of the seeds (40,000 g for 30 min at 20 °C) and the supernatant and pellet fractions analysed by SDS–PAGE, the pellets contained the added seeds (fibrils or ribbons, 10% of the protein), whereas the supernatants contained the soluble α-syn (90% of the protein content of the solution). The amount of α-syn in the pellet and supernatant fractions at the indicated time (2, 24 and 150 h) in the absence (black letters) or the presence of α-syn fibrils (red letters) and ribbons (blue letters) seeds revealed by Coomassie blue stained SDS–PAGE are also shown. The inset in ( a ) corresponds to a blow-up on the initial stages of the assembly reactions. Electron micrographs and SDS–PAGE proteinase K degradation patterns of α-syn assemblies generated upon addition of preformed α-syn fibrils and ribbons are shown. The time (in min) and molecular weight markers (in kDa) are shown on the top and left of each Coomassie blue stained SDS–PAGE. The scale bars in the electron micrographs correspond to 200 nm.
Figure Legend Snippet: Cross-seeding capacities of the two α-syn polymorphs. Elongation of preformed α-syn fibrils (red data points) and ribbons (blue data points), 10 μM in ( a ) buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl), ( b ) buffer B (5 mM Tris-HCl, pH 7.5), at 37 °C, in the presence of soluble α-syn (100 μM), monitored by measurement of scattered light at 440 nm. The control assembly reactions of soluble α-syn in the absence of preformed seeds (black data points) are also shown. When the seeded assembly reactions were centrifuged immediately after addition of the seeds (40,000 g for 30 min at 20 °C) and the supernatant and pellet fractions analysed by SDS–PAGE, the pellets contained the added seeds (fibrils or ribbons, 10% of the protein), whereas the supernatants contained the soluble α-syn (90% of the protein content of the solution). The amount of α-syn in the pellet and supernatant fractions at the indicated time (2, 24 and 150 h) in the absence (black letters) or the presence of α-syn fibrils (red letters) and ribbons (blue letters) seeds revealed by Coomassie blue stained SDS–PAGE are also shown. The inset in ( a ) corresponds to a blow-up on the initial stages of the assembly reactions. Electron micrographs and SDS–PAGE proteinase K degradation patterns of α-syn assemblies generated upon addition of preformed α-syn fibrils and ribbons are shown. The time (in min) and molecular weight markers (in kDa) are shown on the top and left of each Coomassie blue stained SDS–PAGE. The scale bars in the electron micrographs correspond to 200 nm.

Techniques Used: SDS Page, Staining, Generated, Molecular Weight

The two α-syn polymorphs imprint their intrinsic architecture to endogenous α-syn upon its recruitment. Western blot analysis of the degradation profiles of the reporter ChFP-tagged α-syn in Neuro 2A cell lysate (corresponding to a cell density of 4 × 10 6 cell per ml) upon exposure of the cells to exogenous α-syn fibrils or ribbons (2 μM monomeric concentration, for example, particle concentration of 0.25 and 2.2 nM for fibrils and ribbons, respectively) in the presence of the indicated concentrations of proteinase K (μg ml −1 ). The samples were analysed on 15% SDS–PAGE. The degradation profile of ChFP-tagged α-syn puncta seeded by α-syn fibrils ( a ) differs very significantly from that of ChFP-tagged α-syn puncta seeded by α-syn ribbons ( b ). Both patterns differ from that of ChFP-tagged α-syn from untreated Neuro 2A cells ( c ). The immunoreactivity of HSPA8 was used as a loading control ( d ). The molecular mass markers (in kilodaltons) are indicated.
Figure Legend Snippet: The two α-syn polymorphs imprint their intrinsic architecture to endogenous α-syn upon its recruitment. Western blot analysis of the degradation profiles of the reporter ChFP-tagged α-syn in Neuro 2A cell lysate (corresponding to a cell density of 4 × 10 6 cell per ml) upon exposure of the cells to exogenous α-syn fibrils or ribbons (2 μM monomeric concentration, for example, particle concentration of 0.25 and 2.2 nM for fibrils and ribbons, respectively) in the presence of the indicated concentrations of proteinase K (μg ml −1 ). The samples were analysed on 15% SDS–PAGE. The degradation profile of ChFP-tagged α-syn puncta seeded by α-syn fibrils ( a ) differs very significantly from that of ChFP-tagged α-syn puncta seeded by α-syn ribbons ( b ). Both patterns differ from that of ChFP-tagged α-syn from untreated Neuro 2A cells ( c ). The immunoreactivity of HSPA8 was used as a loading control ( d ). The molecular mass markers (in kilodaltons) are indicated.

Techniques Used: Western Blot, Concentration Assay, SDS Page

Structural characterization of the two α-syn polymorphs. ( a ) Time courses of α-syn (100 μM) assembly in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl), red data points, and B (5 mM Tris-HCl, pH 7.5), blue data points, at 37 °C, monitored by measurement of scattered light at 440 nm. ( b ) Time courses of depolymerization at 4 °C of α-syn (100 μM monomer concentration) assemblies obtained in buffer A (high salt, red curve) and B (low salt, blue curve) assessed by quantifying α-syn within the pellet and supernatant fractions by SDS–PAGE, as described in the Methods. Data are mean±s.d. ( n =4). ( c , d ) Negatively stained TEM of α-syn fibrils ( c ) and ribbons ( d ). The arrowheads point to twists; scale bars, 200 nm. ( e , f ), Proteinase K degradation patterns of α-syn (100 μM monomer concentration) fibrils ( e ) and ribbons ( f ), monitored over time on Coomassie stained SDS–PAGE (15%). Time (min) and molecular weight markers (kDa) are shown on the top and left of each gel, respectively. ( g , h ), X-ray diffraction pattern of partially aligned α-syn fibrils ( g ) and ribbons ( h ). α-syn fibrils X-ray scattering pattern is typical of amyloids showing a sharp anisotropic reflection at 4.7 Å along the meridian and another anisotropic reflection at 10 Å along the equator while that of α-syn ribbons resembles a powder/crystalline diffraction pattern with sharp meridional and equatorial reflections at 4.75 and 11 Å as well as long- and short-range reflections. ( i ) Radial averaging of the X-ray scattering patterns of α-syn fibrils (red curve) and ribbons (blue curve). ( j ) The conformational FILA antibody distinguishes equal amounts (0.4 μg) of α-syn fibrils (1) from α-syn ribbons (2) spotted on nitrocellulose membranes, whereas pan-α-syn antibodies (ASY1) do not. ( k ) Backbone Ψ angles as predicted by TALOS for α-syn fibrils (red) and ribbons (blue) based on sequential assignment. Suggested ß-sheet are indicated by arrows, with the lighter colours used for extensions if the glycines are assumed to be part of the ß-sheet or when uncertain TALOS β-sheet predictions (marked by crosses) are included into ß-sheets. Some residues in α-syn fibril show slight peak doubling. This is not considered here, as the resulting backbone angles are very similar.
Figure Legend Snippet: Structural characterization of the two α-syn polymorphs. ( a ) Time courses of α-syn (100 μM) assembly in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl), red data points, and B (5 mM Tris-HCl, pH 7.5), blue data points, at 37 °C, monitored by measurement of scattered light at 440 nm. ( b ) Time courses of depolymerization at 4 °C of α-syn (100 μM monomer concentration) assemblies obtained in buffer A (high salt, red curve) and B (low salt, blue curve) assessed by quantifying α-syn within the pellet and supernatant fractions by SDS–PAGE, as described in the Methods. Data are mean±s.d. ( n =4). ( c , d ) Negatively stained TEM of α-syn fibrils ( c ) and ribbons ( d ). The arrowheads point to twists; scale bars, 200 nm. ( e , f ), Proteinase K degradation patterns of α-syn (100 μM monomer concentration) fibrils ( e ) and ribbons ( f ), monitored over time on Coomassie stained SDS–PAGE (15%). Time (min) and molecular weight markers (kDa) are shown on the top and left of each gel, respectively. ( g , h ), X-ray diffraction pattern of partially aligned α-syn fibrils ( g ) and ribbons ( h ). α-syn fibrils X-ray scattering pattern is typical of amyloids showing a sharp anisotropic reflection at 4.7 Å along the meridian and another anisotropic reflection at 10 Å along the equator while that of α-syn ribbons resembles a powder/crystalline diffraction pattern with sharp meridional and equatorial reflections at 4.75 and 11 Å as well as long- and short-range reflections. ( i ) Radial averaging of the X-ray scattering patterns of α-syn fibrils (red curve) and ribbons (blue curve). ( j ) The conformational FILA antibody distinguishes equal amounts (0.4 μg) of α-syn fibrils (1) from α-syn ribbons (2) spotted on nitrocellulose membranes, whereas pan-α-syn antibodies (ASY1) do not. ( k ) Backbone Ψ angles as predicted by TALOS for α-syn fibrils (red) and ribbons (blue) based on sequential assignment. Suggested ß-sheet are indicated by arrows, with the lighter colours used for extensions if the glycines are assumed to be part of the ß-sheet or when uncertain TALOS β-sheet predictions (marked by crosses) are included into ß-sheets. Some residues in α-syn fibril show slight peak doubling. This is not considered here, as the resulting backbone angles are very similar.

Techniques Used: Concentration Assay, SDS Page, Staining, Transmission Electron Microscopy, Molecular Weight

16) Product Images from "Membrane Insertion of the Bacillus thuringiensis Cry1Ab Toxin: Single Mutation in Domain II Block Partitioning of the Toxin into the Brush Border Membrane †"

Article Title: Membrane Insertion of the Bacillus thuringiensis Cry1Ab Toxin: Single Mutation in Domain II Block Partitioning of the Toxin into the Brush Border Membrane †

Journal: Biochemistry

doi: 10.1021/bi7014234

Steady state fluorescence spectra of Cry1Ab mutants labeled with acrylodan. The samples were excited at 360 nm and the resultant emission recorded from 390 to 650 nm. The relative fluorescence ( y -axis) is expressed in arbitrary units. Correction of the spectra was made against either a buffer blank for the free protein in solution or against SUV or BBMV for the protein bound to the respective vesicles. ( − ) represents the spectra of the purified labeled protein in solution. (· · ·) represents the spectra of the pure labeled protein bound to SUV or BBMV before proteinase K treatment. (---) represents the spectra of labeled protein bound to BBMV or SUV after proteinase K treatment. The y -axis represents the relative fluorescence intensity of each sample under one condition (buffer) to another (in membrane before and after proteinase K) in arbitrary units and may not be interpreted as an absolute value of intensity of fluorescence. A: Cry1AbV171C treated with BBMV. B: Cry1AbF371A/V171C treated with BBMV. C: Cry1AbF371C treated with BBMV. D: Cry1AbV171C treated with SUV. E: Cry1AbF371A/V171C treated with SUV. F: Cry1AbF371C treated with SUV.
Figure Legend Snippet: Steady state fluorescence spectra of Cry1Ab mutants labeled with acrylodan. The samples were excited at 360 nm and the resultant emission recorded from 390 to 650 nm. The relative fluorescence ( y -axis) is expressed in arbitrary units. Correction of the spectra was made against either a buffer blank for the free protein in solution or against SUV or BBMV for the protein bound to the respective vesicles. ( − ) represents the spectra of the purified labeled protein in solution. (· · ·) represents the spectra of the pure labeled protein bound to SUV or BBMV before proteinase K treatment. (---) represents the spectra of labeled protein bound to BBMV or SUV after proteinase K treatment. The y -axis represents the relative fluorescence intensity of each sample under one condition (buffer) to another (in membrane before and after proteinase K) in arbitrary units and may not be interpreted as an absolute value of intensity of fluorescence. A: Cry1AbV171C treated with BBMV. B: Cry1AbF371A/V171C treated with BBMV. C: Cry1AbF371C treated with BBMV. D: Cry1AbV171C treated with SUV. E: Cry1AbF371A/V171C treated with SUV. F: Cry1AbF371C treated with SUV.

Techniques Used: Fluorescence, Labeling, Purification

Proteinase K protection assay of mutants in 3 domains of Cry toxin. Mutations 1Ab S176C (domain I), 1Ab S443C (domain II), and 1Ab F461C (domain III) were expressed and purified (lanes 1, 2, and 3). Proteinase K protection assay was performed, and SDS–PAGE showing a 60 kDa protected form is observed for 1Ab S176C (lane 4), 1Ab S443C (lane 5), and 1Ab F461C (lane 6).
Figure Legend Snippet: Proteinase K protection assay of mutants in 3 domains of Cry toxin. Mutations 1Ab S176C (domain I), 1Ab S443C (domain II), and 1Ab F461C (domain III) were expressed and purified (lanes 1, 2, and 3). Proteinase K protection assay was performed, and SDS–PAGE showing a 60 kDa protected form is observed for 1Ab S176C (lane 4), 1Ab S443C (lane 5), and 1Ab F461C (lane 6).

Techniques Used: Purification, SDS Page

Proteinase K protection assay of Cry1Ab wt and its mutants. Reaction was run on 4–20% SDS–PAGE gels, and the membranes were blotted using anti-1A polyclonal antibody and HRP tagged anti rabbit secondary antibody. Lane1: Pure Cry1Abwt (10 μ g). Lane 2: proteinase K treated BBMV bound to Cry1Ab wt. Lane 3: Pure 1AbV171C (10 μ g). Lane 4: proteinase K treated BBMV bound to 1AbV171C. Lane 5: Pure 1AbF371C (10 μ g). Lane 6: proteinase K treated BBMV bound to 1AbF371C. Lane 7: Pure 1AbF371A/V171C (10 μ g). Lane 8: proteinase K treated BBMV bound to 1AbF371A/V171C.
Figure Legend Snippet: Proteinase K protection assay of Cry1Ab wt and its mutants. Reaction was run on 4–20% SDS–PAGE gels, and the membranes were blotted using anti-1A polyclonal antibody and HRP tagged anti rabbit secondary antibody. Lane1: Pure Cry1Abwt (10 μ g). Lane 2: proteinase K treated BBMV bound to Cry1Ab wt. Lane 3: Pure 1AbV171C (10 μ g). Lane 4: proteinase K treated BBMV bound to 1AbV171C. Lane 5: Pure 1AbF371C (10 μ g). Lane 6: proteinase K treated BBMV bound to 1AbF371C. Lane 7: Pure 1AbF371A/V171C (10 μ g). Lane 8: proteinase K treated BBMV bound to 1AbF371A/V171C.

Techniques Used: SDS Page

17) Product Images from "Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella"

Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-13-80

Expression of OVA-CD4pep-MisL fusion proteins determined by western blot analysis using an anti-OVA-CD4 peptide polyclonal antibody (A) Ovalbumin (lane 1), strain CS4551 with pnirBLTBsp-MisL (lanes 2 and 3), pZS1202 which expresses the (OVA-CD4)-MisL fusion protein from the  nirB  promoter (lanes 4 and 5), or pZS1204 expresses the (OVA-CD4)-MisL fusion protein from the  nirB  and  spiC  promoters in tandem (lanes 6 and 7); (B) Bovine serum albumin (lane 1), ovalbumin (lane 2), untreated (lane 3) or proteinase K-treated (lane 4) outer membrane proteins of  Salmonella  strain SL7207 pZ1204.
Figure Legend Snippet: Expression of OVA-CD4pep-MisL fusion proteins determined by western blot analysis using an anti-OVA-CD4 peptide polyclonal antibody (A) Ovalbumin (lane 1), strain CS4551 with pnirBLTBsp-MisL (lanes 2 and 3), pZS1202 which expresses the (OVA-CD4)-MisL fusion protein from the nirB promoter (lanes 4 and 5), or pZS1204 expresses the (OVA-CD4)-MisL fusion protein from the nirB and spiC promoters in tandem (lanes 6 and 7); (B) Bovine serum albumin (lane 1), ovalbumin (lane 2), untreated (lane 3) or proteinase K-treated (lane 4) outer membrane proteins of Salmonella strain SL7207 pZ1204.

Techniques Used: Expressing, Western Blot

Surface exposure of (OVA-CD4)-MisL fusion protein detected by western blot analysis. Salmonella SL7207 pZS1205 treated with 0 (lane 1), 11 (lane 2) and 33 (lane 3) μg/ml proteinase K and probed with (A) anti-β-lactamase pAb or (B) anti-OVA-CD4 peptide pAb antibodies, respectively. (C) Salmonella SL7207 expressing MisL fusion proteins with one (pZS1205, lanes 1 and 6), two (pZS1205-2, lanes 4 and 7) or four (pZS1205-4, lanes 5 and 8) copies of the OVA-CD4 epitope; untreated (lanes 3 to 5) or treated (lanes 6 to 8) with proteinase K. Bovine albumin (lane 1) and ovalbumin (lane 2), as negative and positive antibody controls. (D) Salmonella SL7207 pgtE expressing MisL fusion proteins with one (pZS1205, lane 1), two (pZS1205-2, lane 2) or four (pZS1205-4, lane 3) copies of the OVA-CD4 epitope.
Figure Legend Snippet: Surface exposure of (OVA-CD4)-MisL fusion protein detected by western blot analysis. Salmonella SL7207 pZS1205 treated with 0 (lane 1), 11 (lane 2) and 33 (lane 3) μg/ml proteinase K and probed with (A) anti-β-lactamase pAb or (B) anti-OVA-CD4 peptide pAb antibodies, respectively. (C) Salmonella SL7207 expressing MisL fusion proteins with one (pZS1205, lanes 1 and 6), two (pZS1205-2, lanes 4 and 7) or four (pZS1205-4, lanes 5 and 8) copies of the OVA-CD4 epitope; untreated (lanes 3 to 5) or treated (lanes 6 to 8) with proteinase K. Bovine albumin (lane 1) and ovalbumin (lane 2), as negative and positive antibody controls. (D) Salmonella SL7207 pgtE expressing MisL fusion proteins with one (pZS1205, lane 1), two (pZS1205-2, lane 2) or four (pZS1205-4, lane 3) copies of the OVA-CD4 epitope.

Techniques Used: Western Blot, Expressing

18) Product Images from "MCU Interacts with Miro1 to Modulate Mitochondrial Functions in Neurons"

Article Title: MCU Interacts with Miro1 to Modulate Mitochondrial Functions in Neurons

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0504-18.2018

The N-terminus of MCU is located on the cytoplasmic side of the outer mitochondrial membrane. A , Coimmunoprecipitation analysis of MCU(Δ2–57)-Flag and Myc-Miro1. MCU(Δ2–57)-Flag was unable to produce the unprocessed 40 kDa band of MCU, and was also unable to coimmunoprecipitate Myc-Miro1. B , C , Colocalization of MCU-Flag and Flag-MCU with Myc-Miro1 in Neuro2A cells. Isolated mitochondria from HEK cells overexpressing Myc-Miro1 and either MCU-Flag or Flag-MCU, showing that Flag-MCU epitope resides in the outer mitochondrial membrane. Scale bar, 1 μm. D , Treatment of mitochondria isolated from HEK cells, and transfected with Flag-MCU, with proteinase K. Proteinase K is able to digest the Flag and TOMM20 signal, while leaving the cytochrome C signal intact. Scale bar, 1 μm. E , Quantification of samples in D . N = 96 mitochondria for TOMM20 control, 51 for TOMM20 proteinase K, 43 for Cytochome C control, 82 for cytochrome C proteinase K.
Figure Legend Snippet: The N-terminus of MCU is located on the cytoplasmic side of the outer mitochondrial membrane. A , Coimmunoprecipitation analysis of MCU(Δ2–57)-Flag and Myc-Miro1. MCU(Δ2–57)-Flag was unable to produce the unprocessed 40 kDa band of MCU, and was also unable to coimmunoprecipitate Myc-Miro1. B , C , Colocalization of MCU-Flag and Flag-MCU with Myc-Miro1 in Neuro2A cells. Isolated mitochondria from HEK cells overexpressing Myc-Miro1 and either MCU-Flag or Flag-MCU, showing that Flag-MCU epitope resides in the outer mitochondrial membrane. Scale bar, 1 μm. D , Treatment of mitochondria isolated from HEK cells, and transfected with Flag-MCU, with proteinase K. Proteinase K is able to digest the Flag and TOMM20 signal, while leaving the cytochrome C signal intact. Scale bar, 1 μm. E , Quantification of samples in D . N = 96 mitochondria for TOMM20 control, 51 for TOMM20 proteinase K, 43 for Cytochome C control, 82 for cytochrome C proteinase K.

Techniques Used: Isolation, Transfection

19) Product Images from "Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation"

Article Title: Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation

Journal: Journal of Extracellular Vesicles

doi: 10.3402/jev.v3.23784

In vitro  uptake of EVs. (A) PC3-EVs were labelled with CellVue Claret and incubated with RAW264.7 or hOB cells at 37°C for the indicated times. Cells were co-stained with anti-tubulin antibody (green staining) and cell surface-bound or cell-internalized EVs were visualized by the red stain. As a control, CellVue dye was centrifuged alongside EVs, and the pellet re-suspended and added to cells (bottom panel). Representative confocal microscopy images are shown. Bar=10 µm. (B, C) EV uptake was quantified using ImageJ to measure total cellular fluorescence for 20–40 cells at each time point, across 3 independent experiments. The background fluorescence of cells incubated with CellVue dye subtracted to derive Δ Mean Fluorescence Intensity (±SEM). (D, E) 20 µg EVs from GFP or cavin-1 PC3 cells were treated with proteinase K, neuraminidase or PBS control, labelled with PKH2 and then incubated with 4×10 5  RAW264.7 cells seeded in tubes at 37°C for 2 hours. Uptake of fluorescence was determined by flow cytometry, with untreated cells used for background fluorescence levels. Data over 3 independent experiments were analysed for (D)% fluorescent cells and (E) Δ Mean Fluorescence Intensity±SEM.
Figure Legend Snippet: In vitro uptake of EVs. (A) PC3-EVs were labelled with CellVue Claret and incubated with RAW264.7 or hOB cells at 37°C for the indicated times. Cells were co-stained with anti-tubulin antibody (green staining) and cell surface-bound or cell-internalized EVs were visualized by the red stain. As a control, CellVue dye was centrifuged alongside EVs, and the pellet re-suspended and added to cells (bottom panel). Representative confocal microscopy images are shown. Bar=10 µm. (B, C) EV uptake was quantified using ImageJ to measure total cellular fluorescence for 20–40 cells at each time point, across 3 independent experiments. The background fluorescence of cells incubated with CellVue dye subtracted to derive Δ Mean Fluorescence Intensity (±SEM). (D, E) 20 µg EVs from GFP or cavin-1 PC3 cells were treated with proteinase K, neuraminidase or PBS control, labelled with PKH2 and then incubated with 4×10 5 RAW264.7 cells seeded in tubes at 37°C for 2 hours. Uptake of fluorescence was determined by flow cytometry, with untreated cells used for background fluorescence levels. Data over 3 independent experiments were analysed for (D)% fluorescent cells and (E) Δ Mean Fluorescence Intensity±SEM.

Techniques Used: In Vitro, Incubation, Staining, Confocal Microscopy, Fluorescence, Flow Cytometry, Cytometry

20) Product Images from "Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment"

Article Title: Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003355

Assembly of the p7 mutants is impaired prior to capsid envelopment. WT-Jc1- or mutant-transfected detergent-free cell lysates were subjected to a proteolytic digestion protection assay as follows. Lysates were separated into three aliquots which received different treatments: (i) left untreated, (ii) treated with 50 µg/ml proteinase K for 1 h on ice, or (iii) lysed in 5% Triton X-100 prior to proteinase K treatment (condition used for background correction). The amount of protease-resistant core was determined by Western Blot and ELISA. (A) Representative Western Blot stained for HCV core. (B) Western Blot signal intensities were quantified with LabImage 1D and values obtained for the proteinase K-treated sample were background-corrected and normalized to untreated control. Mean values and standard deviations of 3–6 independent experiments are shown.
Figure Legend Snippet: Assembly of the p7 mutants is impaired prior to capsid envelopment. WT-Jc1- or mutant-transfected detergent-free cell lysates were subjected to a proteolytic digestion protection assay as follows. Lysates were separated into three aliquots which received different treatments: (i) left untreated, (ii) treated with 50 µg/ml proteinase K for 1 h on ice, or (iii) lysed in 5% Triton X-100 prior to proteinase K treatment (condition used for background correction). The amount of protease-resistant core was determined by Western Blot and ELISA. (A) Representative Western Blot stained for HCV core. (B) Western Blot signal intensities were quantified with LabImage 1D and values obtained for the proteinase K-treated sample were background-corrected and normalized to untreated control. Mean values and standard deviations of 3–6 independent experiments are shown.

Techniques Used: Mutagenesis, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Characterization of differentially-sedimenting core complexes. Lysates of cells transfected with WT or mutant Jc1 were separated by rate zonal density gradient centrifugation. Each fraction was analyzed for infectivity by TCID 50  (A), core protein content by ELISA (B), and RNA copy number by qRT-PCR (C). Fractions 4 and 7 are highlighted with black arrows. (D) Fractions 4 and 7 were subjected to a proteolytic digestion protection assay and the amount of protease-resistant core protein was quantified by ELISA. The values obtained for the proteinase K-treated samples were background-subtracted and normalized to the untreated controls. Mean values and standard deviations of 2 independent experiments are shown. (E) Lysates of WT Jc1-transfected cells were separated by rate zonal density gradient centrifugation in the absence or presence of 1% DDM and core distribution along the gradient was determined by ELISA.
Figure Legend Snippet: Characterization of differentially-sedimenting core complexes. Lysates of cells transfected with WT or mutant Jc1 were separated by rate zonal density gradient centrifugation. Each fraction was analyzed for infectivity by TCID 50 (A), core protein content by ELISA (B), and RNA copy number by qRT-PCR (C). Fractions 4 and 7 are highlighted with black arrows. (D) Fractions 4 and 7 were subjected to a proteolytic digestion protection assay and the amount of protease-resistant core protein was quantified by ELISA. The values obtained for the proteinase K-treated samples were background-subtracted and normalized to the untreated controls. Mean values and standard deviations of 2 independent experiments are shown. (E) Lysates of WT Jc1-transfected cells were separated by rate zonal density gradient centrifugation in the absence or presence of 1% DDM and core distribution along the gradient was determined by ELISA.

Techniques Used: Transfection, Mutagenesis, Gradient Centrifugation, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Mutations in p7 affect capsid assembly. (A) WT-Jc1 or mutant-transfected cells were harvested 48 h post-transfection. Cell lysates were subjected to rate zonal density gradient centrifugation in the presence of non-ionic detergent (1% DDM) and core protein amounts along the gradient were measured by ELISA. Fractions 3 to 5 are highlighted with black arrows. The right panel shows the core amount relative to total core expression for fraction 5. (B) RNase digestion protection assay (see Materials and Methods ). Fractions 3 to 5 of the WT-Jc1 gradient were subjected to RNase digestion or left untreated. As a background control, samples were treated with proteinase K to remove protecting capsids, prior to RNase treatment. The amount of residual HCV RNA was quantified by qRT-PCR. In parallel crude HCV replication complexes (CRCs) were prepared by the method described by Quinkert et al. [62] . CRCs were then subjected to RNase treatment as described above. To mimic conditions of the sedimentation 1% DDM was added. (C) Separation of core complexes by blue-native-PAGE (4–16% gradient gel). (D) Two-dimensional gel electrophoresis of core complexes. Cell lysates were separated by blue native-PAGE (3–12% gradient gel) in a first electrophoresis and by SDS-PAGE in the second dimension. The results of three independent experiments are shown side by side. Gel pictures are shown vertically for easier comparison with the blue-native-PAGE results. (C, D) Core protein was detected by Western Blotting and subsequent immunodetection with the core-specific monoclonal antibody C7-50.
Figure Legend Snippet: Mutations in p7 affect capsid assembly. (A) WT-Jc1 or mutant-transfected cells were harvested 48 h post-transfection. Cell lysates were subjected to rate zonal density gradient centrifugation in the presence of non-ionic detergent (1% DDM) and core protein amounts along the gradient were measured by ELISA. Fractions 3 to 5 are highlighted with black arrows. The right panel shows the core amount relative to total core expression for fraction 5. (B) RNase digestion protection assay (see Materials and Methods ). Fractions 3 to 5 of the WT-Jc1 gradient were subjected to RNase digestion or left untreated. As a background control, samples were treated with proteinase K to remove protecting capsids, prior to RNase treatment. The amount of residual HCV RNA was quantified by qRT-PCR. In parallel crude HCV replication complexes (CRCs) were prepared by the method described by Quinkert et al. [62] . CRCs were then subjected to RNase treatment as described above. To mimic conditions of the sedimentation 1% DDM was added. (C) Separation of core complexes by blue-native-PAGE (4–16% gradient gel). (D) Two-dimensional gel electrophoresis of core complexes. Cell lysates were separated by blue native-PAGE (3–12% gradient gel) in a first electrophoresis and by SDS-PAGE in the second dimension. The results of three independent experiments are shown side by side. Gel pictures are shown vertically for easier comparison with the blue-native-PAGE results. (C, D) Core protein was detected by Western Blotting and subsequent immunodetection with the core-specific monoclonal antibody C7-50.

Techniques Used: Mutagenesis, Transfection, Gradient Centrifugation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Sedimentation, Blue Native PAGE, Two-Dimensional Gel Electrophoresis, Electrophoresis, SDS Page, Western Blot, Immunodetection

21) Product Images from "Changes in Lipopolysaccharide O Antigen Distinguish Acute versus Chronic Leptospira interrogans Infections "

Article Title: Changes in Lipopolysaccharide O Antigen Distinguish Acute versus Chronic Leptospira interrogans Infections

Journal: Infection and Immunity

doi: 10.1128/IAI.73.6.3251-3260.2005

Antigenic composition of guinea pig liver-derived HTL and demonstration that the content of a proteinase K-resistant 22-kDa antigen is diminished. Immunoblots of equal numbers of whole IVCL cells purified over Percoll (lane 1), whole guinea pig liver-derived HTL cells purified over Percoll (lane 2), Percoll-purified IVCL cells treated with proteinase K (lane 3), and Percoll-purified HTL cells treated with proteinase K (lane 4) were probed with either antiserum specific for OMV of Leptospira ) (D) to verify that similar amounts of Leptospira cells were loaded in each lane. The integrated density values (IDV) demonstrate that the diminished content of the 22-kDa proteinase K-resistant antigen was not due to smaller amounts of HTL being compared to IVCL. The positions of molecular mass markers (in kilodaltons) are indicated to the left of each blot. An star indicates the position of the 22-kDa antigen expressed in smaller amounts in HTL than in IVCL.
Figure Legend Snippet: Antigenic composition of guinea pig liver-derived HTL and demonstration that the content of a proteinase K-resistant 22-kDa antigen is diminished. Immunoblots of equal numbers of whole IVCL cells purified over Percoll (lane 1), whole guinea pig liver-derived HTL cells purified over Percoll (lane 2), Percoll-purified IVCL cells treated with proteinase K (lane 3), and Percoll-purified HTL cells treated with proteinase K (lane 4) were probed with either antiserum specific for OMV of Leptospira ) (D) to verify that similar amounts of Leptospira cells were loaded in each lane. The integrated density values (IDV) demonstrate that the diminished content of the 22-kDa proteinase K-resistant antigen was not due to smaller amounts of HTL being compared to IVCL. The positions of molecular mass markers (in kilodaltons) are indicated to the left of each blot. An star indicates the position of the 22-kDa antigen expressed in smaller amounts in HTL than in IVCL.

Techniques Used: Derivative Assay, Western Blot, Purification

Reduced Oag content in guinea pig liver-derived HTL compared to IVCL. Parallel immunoblots of proteinase K-treated Percoll-purified IVCL (lane 1) and HTL (lane 2) were probed with monoclonal antibody F70C24 (A) or CRS (B). In both cases the diminution in Oag content of HTL is apparent. (C) Periodate-silver staining of whole IVCL (lane 1) and HTL (lane 2), proteinase K-treated IVCL (lane 3), and proteinase K-treated HTL (lane 4) shows that no 22-kDa Oag band is seen in proteinase K-treated HTL.
Figure Legend Snippet: Reduced Oag content in guinea pig liver-derived HTL compared to IVCL. Parallel immunoblots of proteinase K-treated Percoll-purified IVCL (lane 1) and HTL (lane 2) were probed with monoclonal antibody F70C24 (A) or CRS (B). In both cases the diminution in Oag content of HTL is apparent. (C) Periodate-silver staining of whole IVCL (lane 1) and HTL (lane 2), proteinase K-treated IVCL (lane 3), and proteinase K-treated HTL (lane 4) shows that no 22-kDa Oag band is seen in proteinase K-treated HTL.

Techniques Used: Derivative Assay, Western Blot, Purification, Silver Staining

22) Product Images from "Prion Pathogenesis in the Absence of NLRP3/ASC Inflammasomes"

Article Title: Prion Pathogenesis in the Absence of NLRP3/ASC Inflammasomes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117208

Prion disease in the absence of NLRP3. A-B  Kaplan-Meier survival plots of  Nlrp3 -/-  (blue line) and C57BL/6 wild-type mice (black line) inoculated intracerebrally with 90 ng (A) or 30 ng of RML6 (B). For each experimental group, the number of mice is indicated (n). Censored events (ticks) indicate intercurrent deaths not related with prion disease. With both doses, no statistically significant difference was observed (90 ng = median survival  Nlrp3 -/-  182 dpi, C57BL/6 181 dpi, P = 0.79; 30 ng =  Nlrp3 -/-  211 dpi, C57BL/6 206 dpi, P = 0.21, log-rank test).  C  Histological analysis of  Nlrp3 -/-  (I-IV) and C57BL/6 wild-type mice (V-VIII). Representative hematoxylin and eosin staining (H  E) and immunohistochemical stainings for microglia (IBA1), astrocytes (GFAP) and partially protease-resistant PrP (SAF-84) in cerebellar areas are displayed. Scale bar: 250 µm.  D  Western blotting analysis of partially protease K (PK)-resistant PrP as detected with POM1 in brain homogenates after PK digestion (PK +). Neg Ctrl: brain homogenate from a control mouse not inoculated with prions is shown with (PK +) and without (PK -) PK digestion.  E  Levels of IL-1β in brains of terminally sick mice. Each point denotes one mouse. Mean +/- standard deviation are shown. No significant difference was observed (P = 0.13, Student’s t test). C, D and E are referred to mice inoculated with 30 ng of RML6.
Figure Legend Snippet: Prion disease in the absence of NLRP3. A-B Kaplan-Meier survival plots of Nlrp3 -/- (blue line) and C57BL/6 wild-type mice (black line) inoculated intracerebrally with 90 ng (A) or 30 ng of RML6 (B). For each experimental group, the number of mice is indicated (n). Censored events (ticks) indicate intercurrent deaths not related with prion disease. With both doses, no statistically significant difference was observed (90 ng = median survival Nlrp3 -/- 182 dpi, C57BL/6 181 dpi, P = 0.79; 30 ng = Nlrp3 -/- 211 dpi, C57BL/6 206 dpi, P = 0.21, log-rank test). C Histological analysis of Nlrp3 -/- (I-IV) and C57BL/6 wild-type mice (V-VIII). Representative hematoxylin and eosin staining (H E) and immunohistochemical stainings for microglia (IBA1), astrocytes (GFAP) and partially protease-resistant PrP (SAF-84) in cerebellar areas are displayed. Scale bar: 250 µm. D Western blotting analysis of partially protease K (PK)-resistant PrP as detected with POM1 in brain homogenates after PK digestion (PK +). Neg Ctrl: brain homogenate from a control mouse not inoculated with prions is shown with (PK +) and without (PK -) PK digestion. E Levels of IL-1β in brains of terminally sick mice. Each point denotes one mouse. Mean +/- standard deviation are shown. No significant difference was observed (P = 0.13, Student’s t test). C, D and E are referred to mice inoculated with 30 ng of RML6.

Techniques Used: Mouse Assay, Staining, Immunohistochemistry, Western Blot, Standard Deviation

Prion disease in the absence of ASC. A-B  Kaplan-Meier survival plots of  Pycard -/-  (red line) and C57BL/6 wild-type mice (black line) inoculated intracerebrally with 90 ng (A) or 30 ng of RML6 prions (B). For each experimental group, the number of mice is indicated (n). With both doses, no statistically significant difference was observed (90 ng = median survival  Pycard -/-  189 dpi, C57BL/6 194 dpi, P = 0.35; 30 ng =  Pycard -/-  187 dpi, C57BL/6 191 dpi, P = 0.23, log-rank test).  C  Histological analysis of  Pycard -/-  (I-IV) and C57BL/6 wild-type mice (V-VIII). Representative hematoxylin and eosin staining (H  E) and immunohistochemical stainings for microglia (IBA1), astrocytes (GFAP) and partially protease-resistant PrP (SAF-84) in cerebellar areas are displayed. Scale bar: 250 µm.  D  Western blotting analysis of partially protease K (PK)-resistant PrP as detected with POM1 in brain homogenates after PK digestion (PK +). Neg Ctrl: brain homogenate from a control mouse not inoculated with prions is shown with (PK +) and without (PK -) PK digestion.  E  Levels of IL-1β in brains of terminally sick mice. Each point denotes one mouse. Mean +/- standard deviation are shown. No significant difference was observed (P = 0.28, Student’s t test). C, D and E are referred to mice inoculated with 30 ng of RML6.
Figure Legend Snippet: Prion disease in the absence of ASC. A-B Kaplan-Meier survival plots of Pycard -/- (red line) and C57BL/6 wild-type mice (black line) inoculated intracerebrally with 90 ng (A) or 30 ng of RML6 prions (B). For each experimental group, the number of mice is indicated (n). With both doses, no statistically significant difference was observed (90 ng = median survival Pycard -/- 189 dpi, C57BL/6 194 dpi, P = 0.35; 30 ng = Pycard -/- 187 dpi, C57BL/6 191 dpi, P = 0.23, log-rank test). C Histological analysis of Pycard -/- (I-IV) and C57BL/6 wild-type mice (V-VIII). Representative hematoxylin and eosin staining (H E) and immunohistochemical stainings for microglia (IBA1), astrocytes (GFAP) and partially protease-resistant PrP (SAF-84) in cerebellar areas are displayed. Scale bar: 250 µm. D Western blotting analysis of partially protease K (PK)-resistant PrP as detected with POM1 in brain homogenates after PK digestion (PK +). Neg Ctrl: brain homogenate from a control mouse not inoculated with prions is shown with (PK +) and without (PK -) PK digestion. E Levels of IL-1β in brains of terminally sick mice. Each point denotes one mouse. Mean +/- standard deviation are shown. No significant difference was observed (P = 0.28, Student’s t test). C, D and E are referred to mice inoculated with 30 ng of RML6.

Techniques Used: Mouse Assay, Staining, Immunohistochemistry, Western Blot, Standard Deviation

23) Product Images from "Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice"

Article Title: Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice

Journal: Veterinary Research

doi: 10.1186/s13567-015-0211-2

Western blot analysis of proteinase-K resistant PrP Sc analyzed using monoclonal antibody T2. A PrP Sc in the brain (Br) and spleen (Sp) of wild-type mice inoculated with sheep-passaged L-BSE at the first and second passage. All samples were digested with 50 μg/mL of proteinase-K at 37 °C for 1 h. Lanes from left to right were loaded with 0.625, 5, 0.0125, and 0.36 mg tissue equivalent, respectively. The molecular markers are shown on the left (kDa). B PrP Sc in the brain of C-BSE- and L-BSE-affected cattle and mice. Lane 1: C-BSE affected cattle, Lane 2: C-BSE affected ICR mouse, Lane 3: L-BSE affected cattle, Lane 4: L-BSE affected sheep, and Lane 5: sheep-passaged L-BSE affected ICR mouse at second passage. Lanes 1, 3, and 4, and Lanes 2 and 5 were loaded with 1.25 and 0.125 mg tissue equivalent, respectively. C Quantification of the relative amounts of the di-, mono-, and unglycosylated forms of PrP Sc from the brain. The column numbers are as listed in ( B ). Bar diagram indicates the diglycosylated form (black), monoglycosylated form (gray), and unglycosylated form (white). Data are expressed as mean ± standard deviation of triplicate experiments.
Figure Legend Snippet: Western blot analysis of proteinase-K resistant PrP Sc analyzed using monoclonal antibody T2. A PrP Sc in the brain (Br) and spleen (Sp) of wild-type mice inoculated with sheep-passaged L-BSE at the first and second passage. All samples were digested with 50 μg/mL of proteinase-K at 37 °C for 1 h. Lanes from left to right were loaded with 0.625, 5, 0.0125, and 0.36 mg tissue equivalent, respectively. The molecular markers are shown on the left (kDa). B PrP Sc in the brain of C-BSE- and L-BSE-affected cattle and mice. Lane 1: C-BSE affected cattle, Lane 2: C-BSE affected ICR mouse, Lane 3: L-BSE affected cattle, Lane 4: L-BSE affected sheep, and Lane 5: sheep-passaged L-BSE affected ICR mouse at second passage. Lanes 1, 3, and 4, and Lanes 2 and 5 were loaded with 1.25 and 0.125 mg tissue equivalent, respectively. C Quantification of the relative amounts of the di-, mono-, and unglycosylated forms of PrP Sc from the brain. The column numbers are as listed in ( B ). Bar diagram indicates the diglycosylated form (black), monoglycosylated form (gray), and unglycosylated form (white). Data are expressed as mean ± standard deviation of triplicate experiments.

Techniques Used: Western Blot, Mouse Assay, Standard Deviation

Western blot analysis of proteinase-K resistant L-BSE PrP Sc in TgBoPrP mice before and after passage in sheep. The brain samples of TgBoPrP mice inoculated intracerebrally with either L-BSE/cattle or L-BSE/sheep were analyzed. PrP Sc in the twice-passaged mice was detected with mAb T2. The numbers indicate three different mice.
Figure Legend Snippet: Western blot analysis of proteinase-K resistant L-BSE PrP Sc in TgBoPrP mice before and after passage in sheep. The brain samples of TgBoPrP mice inoculated intracerebrally with either L-BSE/cattle or L-BSE/sheep were analyzed. PrP Sc in the twice-passaged mice was detected with mAb T2. The numbers indicate three different mice.

Techniques Used: Western Blot, Mouse Assay

24) Product Images from "Important but Differential Roles for Actin in Trafficking of Epstein-Barr Virus in B Cells and Epithelial Cells"

Article Title: Important but Differential Roles for Actin in Trafficking of Epstein-Barr Virus in B Cells and Epithelial Cells

Journal: Journal of Virology

doi: 10.1128/JVI.05883-11

Virus DNA in the nucleus of an SVKCR2 epithelial cell remains stable. (A) Western blot of isolated nuclear and cytoplasmic fractions stained for α-tubulin or lamin B. (B) Virus was bound on ice for 2 h, unbound virus was removed, and cells were warmed for the times indicated. Surface-bound virus was removed by digestion with proteinase K. The total amount of virus DNA remaining per cell was compared with the amount in the fractionated nucleus by QPCR. Error bars are the standard deviations of triplicates.
Figure Legend Snippet: Virus DNA in the nucleus of an SVKCR2 epithelial cell remains stable. (A) Western blot of isolated nuclear and cytoplasmic fractions stained for α-tubulin or lamin B. (B) Virus was bound on ice for 2 h, unbound virus was removed, and cells were warmed for the times indicated. Surface-bound virus was removed by digestion with proteinase K. The total amount of virus DNA remaining per cell was compared with the amount in the fractionated nucleus by QPCR. Error bars are the standard deviations of triplicates.

Techniques Used: Western Blot, Isolation, Staining, Real-time Polymerase Chain Reaction

Virus DNA is lost following internalization into an epithelial cell but not a B cell. Virus was bound on ice for 2 h to Akata and Raji B cells, SVKCR2 and AGS epithelial cells, primary tonsil epithelial cells, or SVKCR2 CIITA cells which express HLA class II. Unbound virus was removed, and cells were warmed for the times indicated. Surface-bound virus was removed by digestion with proteinase K, and virus (BamK) and cellular (CRP) copies were measured by QPCR. Error bars are the standard deviations of triplicates.
Figure Legend Snippet: Virus DNA is lost following internalization into an epithelial cell but not a B cell. Virus was bound on ice for 2 h to Akata and Raji B cells, SVKCR2 and AGS epithelial cells, primary tonsil epithelial cells, or SVKCR2 CIITA cells which express HLA class II. Unbound virus was removed, and cells were warmed for the times indicated. Surface-bound virus was removed by digestion with proteinase K, and virus (BamK) and cellular (CRP) copies were measured by QPCR. Error bars are the standard deviations of triplicates.

Techniques Used: Real-time Polymerase Chain Reaction

Inhibitors of actin remodeling prevent virus internalization into a B cell but enhance internalization into an epithelial cell. Drugs that influence the microtubule network have no effect. (Top panel) Akata B cells, LCL, and SVKCR2, AGS, or NOK epithelial cells were pretreated for 1 h with 5 μM latrunculin A (Lat A) or 0.5 μM jasplakinolide (Jas), virus was bound on ice for 2 h, and cells and bound virus were warmed for 4 h in the presence of drug. Surface-bound virus was removed by proteinase K digestion, and the change in virus DNA copies remaining per cell was measured by QPCR. The horizontal line at 1 indicates the virus DNA copies remaining per cell in the absence of drug. (Bottom panels) Akata B cells or SVKCR2 epithelial cells were pretreated with 10 μM nocodazole (Noco) or 20 μM paclitaxel for 1 h, virus was bound on ice for 2 h, and cells and bound virus were warmed for 4 h in the presence of drug. Surface-bound virus was removed by proteinase K digestion, and the change in virus DNA copies remaining per cell was measured by QPCR. Error bars are the standard deviations of triplicates.
Figure Legend Snippet: Inhibitors of actin remodeling prevent virus internalization into a B cell but enhance internalization into an epithelial cell. Drugs that influence the microtubule network have no effect. (Top panel) Akata B cells, LCL, and SVKCR2, AGS, or NOK epithelial cells were pretreated for 1 h with 5 μM latrunculin A (Lat A) or 0.5 μM jasplakinolide (Jas), virus was bound on ice for 2 h, and cells and bound virus were warmed for 4 h in the presence of drug. Surface-bound virus was removed by proteinase K digestion, and the change in virus DNA copies remaining per cell was measured by QPCR. The horizontal line at 1 indicates the virus DNA copies remaining per cell in the absence of drug. (Bottom panels) Akata B cells or SVKCR2 epithelial cells were pretreated with 10 μM nocodazole (Noco) or 20 μM paclitaxel for 1 h, virus was bound on ice for 2 h, and cells and bound virus were warmed for 4 h in the presence of drug. Surface-bound virus was removed by proteinase K digestion, and the change in virus DNA copies remaining per cell was measured by QPCR. Error bars are the standard deviations of triplicates.

Techniques Used: Real-time Polymerase Chain Reaction

25) Product Images from "Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons"

Article Title: Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons

Journal: PLoS ONE

doi: 10.1371/journal.pone.0119863

Western blot of prion protein in brain of hamsters following intranasal inoculation of DY TME agent in the absence and presence of a preexisting lesion to the olfactory epithelium. Brain from hamsters at sacrifice (see Table 1 ) from vehicle (A) and methimazole (B) treatment groups followed by intranasal inoculation of DY TME were enriched for PrP Sc by detergent extraction, differential ultracentrifugation, and proteinase K digestion. For each sample 20 mg tissue equivalents was examined by Western blot for PrP Sc , except for two samples in which only 2 mg tissue equivalents was used (B, lanes 1 and 5). None of the hamsters in the vehicle group and only one hamster in the methimazole group exhibited symptoms of DY TME (B, lane 5 and Table 1 ). The hamster exhibiting clinical symptoms had a strong PrP Sc signal in brain, and two additional hamsters in the methimazole group that were clinically normal and sacrificed after 414 days postinoculation also had evidence of PrP Sc in brain (B, lanes 1 and 7). Molecular weight markers at edge of western blots correspond to 20, 30, and 40 kilodaltons.
Figure Legend Snippet: Western blot of prion protein in brain of hamsters following intranasal inoculation of DY TME agent in the absence and presence of a preexisting lesion to the olfactory epithelium. Brain from hamsters at sacrifice (see Table 1 ) from vehicle (A) and methimazole (B) treatment groups followed by intranasal inoculation of DY TME were enriched for PrP Sc by detergent extraction, differential ultracentrifugation, and proteinase K digestion. For each sample 20 mg tissue equivalents was examined by Western blot for PrP Sc , except for two samples in which only 2 mg tissue equivalents was used (B, lanes 1 and 5). None of the hamsters in the vehicle group and only one hamster in the methimazole group exhibited symptoms of DY TME (B, lane 5 and Table 1 ). The hamster exhibiting clinical symptoms had a strong PrP Sc signal in brain, and two additional hamsters in the methimazole group that were clinically normal and sacrificed after 414 days postinoculation also had evidence of PrP Sc in brain (B, lanes 1 and 7). Molecular weight markers at edge of western blots correspond to 20, 30, and 40 kilodaltons.

Techniques Used: Western Blot, Molecular Weight

Western blot of prion protein in brain, olfactory bulb, and nasal turbinate of mice following intranasal inoculation of prions in the absence and presence of a preexisting lesion to the olfactory epithelium. Immunodetection of the prion protein in tissues from C57Bl/6 mice (A, B) and HPrP7752KO transgenic mice (C, D) following intranasal inoculation of RML scrapie and HY TME, respectively. Mice were pretreated with vehicle (veh) and methimazole (mmi) 48 hours prior to prion inoculation. Clinically ill C57Bl/6 mice all had PrP Sc deposition in brain (not shown), olfactory bulb (A), and nasal turbinate (B) following proteinase K (PK) digestion of tissue homogenates (A) and PrP Sc enrichment methods that included a PK digestion step (B). In clinically ill HPrP7752KO mice, PrP Sc was detected in brain (C), olfactory bulb (not shown), and in > 75% of nasal turbinates (D) following PrP Sc enrichment. Asterisk (C, D) indicates a mouse that did not develop clinical symptoms of prion disease and was devoid of PrP Sc . In non-proteinase K (non-PK) treated samples, 20 μg protein from brain (C) and 40 μg protein from nasal turbinate (D) were analyzed while for PK treated samples, 100 μg protein from brain (C) and 1 mg protein from nasal turbinate (D) were used as starting material for PrP Sc enrichment and analysis. RML scrapie-infected brain (Br) control is indicated.
Figure Legend Snippet: Western blot of prion protein in brain, olfactory bulb, and nasal turbinate of mice following intranasal inoculation of prions in the absence and presence of a preexisting lesion to the olfactory epithelium. Immunodetection of the prion protein in tissues from C57Bl/6 mice (A, B) and HPrP7752KO transgenic mice (C, D) following intranasal inoculation of RML scrapie and HY TME, respectively. Mice were pretreated with vehicle (veh) and methimazole (mmi) 48 hours prior to prion inoculation. Clinically ill C57Bl/6 mice all had PrP Sc deposition in brain (not shown), olfactory bulb (A), and nasal turbinate (B) following proteinase K (PK) digestion of tissue homogenates (A) and PrP Sc enrichment methods that included a PK digestion step (B). In clinically ill HPrP7752KO mice, PrP Sc was detected in brain (C), olfactory bulb (not shown), and in > 75% of nasal turbinates (D) following PrP Sc enrichment. Asterisk (C, D) indicates a mouse that did not develop clinical symptoms of prion disease and was devoid of PrP Sc . In non-proteinase K (non-PK) treated samples, 20 μg protein from brain (C) and 40 μg protein from nasal turbinate (D) were analyzed while for PK treated samples, 100 μg protein from brain (C) and 1 mg protein from nasal turbinate (D) were used as starting material for PrP Sc enrichment and analysis. RML scrapie-infected brain (Br) control is indicated.

Techniques Used: Western Blot, Mouse Assay, Immunodetection, Transgenic Assay, Infection

26) Product Images from "Proteomic analysis of purified turkey adenovirus 3 virions"

Article Title: Proteomic analysis of purified turkey adenovirus 3 virions

Journal: Veterinary Research

doi: 10.1186/s13567-015-0214-z

Identification of viral and host virion proteins.  Venn diagrams of viral ( A ) and host proteins ( B ) detected in TAdV-3 untreated and proteinase K-treated samples by in-solution tryptic digestion followed by analysis using 1D-liquid chromatography combined with a mass spectrometer (LC-MS/MS).  C  Eighteen host-incorporated proteins identified in purified proteinase treated TAdV-3 virions in the presence of proteolytic digestion are classified based on their known functions.
Figure Legend Snippet: Identification of viral and host virion proteins. Venn diagrams of viral ( A ) and host proteins ( B ) detected in TAdV-3 untreated and proteinase K-treated samples by in-solution tryptic digestion followed by analysis using 1D-liquid chromatography combined with a mass spectrometer (LC-MS/MS). C Eighteen host-incorporated proteins identified in purified proteinase treated TAdV-3 virions in the presence of proteolytic digestion are classified based on their known functions.

Techniques Used: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Purification

Western blot analysis of host proteins in TAdV-3. Proteins from the proteinase K untreated purified TAdV-3 virions (panels A , B , lane 1) and proteinase K (20 μg incubated in 1 mL of MNT buffer) treated purified TAdV-3 virions (panels A , B , lane 2) were separated by 10–15% SDS-PAGE, transferred to nitrocellulose and analyzed by Western blot using anti-collagen alpha-1(VI) chain serum (panel A ) and anti-haemoglobin serum (panel B ). Molecular weight markers (Lane M).
Figure Legend Snippet: Western blot analysis of host proteins in TAdV-3. Proteins from the proteinase K untreated purified TAdV-3 virions (panels A , B , lane 1) and proteinase K (20 μg incubated in 1 mL of MNT buffer) treated purified TAdV-3 virions (panels A , B , lane 2) were separated by 10–15% SDS-PAGE, transferred to nitrocellulose and analyzed by Western blot using anti-collagen alpha-1(VI) chain serum (panel A ) and anti-haemoglobin serum (panel B ). Molecular weight markers (Lane M).

Techniques Used: Western Blot, Purification, Incubation, SDS Page, Molecular Weight

Proteinase K digestion of purified TAdV-3 virions. A Proteins from purified TAdV-3 untreated (lane 1) or treated (lanes 2–5) with indicated amounts of proteinase K were separated by 10–15% SDS-PAGE, transferred to nitrocellulose and probed by Western blot using anti-TAdV-3 serum. The hexon protein and the fiber protein are depicted by an arrow. Concentration of proteinase K in μg is indicated on top of the panels. B Purified TAdV-3 treated with 20 μg of proteinase K were negatively stained with 2% aqueous phosphotungstic acid and analyzed by transmission electron microscopy. (Direct magnification 50000×, left panel) and (direct magnification 150000×, right panel).
Figure Legend Snippet: Proteinase K digestion of purified TAdV-3 virions. A Proteins from purified TAdV-3 untreated (lane 1) or treated (lanes 2–5) with indicated amounts of proteinase K were separated by 10–15% SDS-PAGE, transferred to nitrocellulose and probed by Western blot using anti-TAdV-3 serum. The hexon protein and the fiber protein are depicted by an arrow. Concentration of proteinase K in μg is indicated on top of the panels. B Purified TAdV-3 treated with 20 μg of proteinase K were negatively stained with 2% aqueous phosphotungstic acid and analyzed by transmission electron microscopy. (Direct magnification 50000×, left panel) and (direct magnification 150000×, right panel).

Techniques Used: Purification, SDS Page, Western Blot, Concentration Assay, Staining, Transmission Assay, Electron Microscopy

27) Product Images from "Complement Regulatory Protein Factor H Is a Soluble Prion Receptor That Potentiates Peripheral Prion Pathogenesis"

Article Title: Complement Regulatory Protein Factor H Is a Soluble Prion Receptor That Potentiates Peripheral Prion Pathogenesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1701100

Factor H directly interacts with prion amyloid enriched from infected brain (A) Prion rods were enriched from infected brains as previously described and (B) are protease K (PK) resistant. The line to the left of the blot marks 25 kD molecular weight. (C) Factor H biochemically interacts with prion rods of both HY and DY strains manually coupled to a surface plasmon resonance sensor chip. (D) Affinity analyses show that fH bound HY prions with slightly higher affinity than DY prions. Traces from the bottom to top of the sensograms show binding of 0 nM, 5 nM, 10 nM, 100 nM, 100 nM, 500 nM, 1000 nM, and 2000 nM fH analytes to prions coated on the chip.
Figure Legend Snippet: Factor H directly interacts with prion amyloid enriched from infected brain (A) Prion rods were enriched from infected brains as previously described and (B) are protease K (PK) resistant. The line to the left of the blot marks 25 kD molecular weight. (C) Factor H biochemically interacts with prion rods of both HY and DY strains manually coupled to a surface plasmon resonance sensor chip. (D) Affinity analyses show that fH bound HY prions with slightly higher affinity than DY prions. Traces from the bottom to top of the sensograms show binding of 0 nM, 5 nM, 10 nM, 100 nM, 100 nM, 500 nM, 1000 nM, and 2000 nM fH analytes to prions coated on the chip.

Techniques Used: Infection, Molecular Weight, SPR Assay, Chromatin Immunoprecipitation, Binding Assay

Factor H promotes early prion propagation in spleen at early timepoints Mice (n ≥ 6 mice/genotype of both sexes) were sacrificed at 30, 60, 90 dpi, and 120 dpi, and their spleens (10% w/v homogenates) were first treated with proteinase K (PK, 10 μg/mL, panel A). Lines to the left of each blot mark 25 kD molecular weight. RML infected animals negative for PrP Sc after straight western blotting (A) were subjected to serial rounds of PMCA (B,C). Asterisks indicate samples scored as positive for PrP Sc . Each biological replicate was run in technical duplicates, and each lane represents one mouse. Western blot analysis of PMCA material (B) shows a higher proportion of RML-infected wild type mice (solid line) positive by round 1 of PMCA, (top blot), whereas most knockout mice (bottom blot, dotted line) required 2 or more rounds of PMCA to visualize PrP Sc . RML (far right) represents 0.05% RML5 amplified prions and serves as a positive control for each PMCA round. ANOVA analysis revealed no significant differences at 30 dpi, but significant differences between the genotypes at 60, 90, and 120 dpi ((p=0.0236, 0.0021, and 0.0032, respectively). Samples in first lane of each blot were not PK digested. Numbers > 100 rpu in the grey area in graph (C) represent scores from samples detected without amplification, and therefore fall outside the dynamic range of PMCA.
Figure Legend Snippet: Factor H promotes early prion propagation in spleen at early timepoints Mice (n ≥ 6 mice/genotype of both sexes) were sacrificed at 30, 60, 90 dpi, and 120 dpi, and their spleens (10% w/v homogenates) were first treated with proteinase K (PK, 10 μg/mL, panel A). Lines to the left of each blot mark 25 kD molecular weight. RML infected animals negative for PrP Sc after straight western blotting (A) were subjected to serial rounds of PMCA (B,C). Asterisks indicate samples scored as positive for PrP Sc . Each biological replicate was run in technical duplicates, and each lane represents one mouse. Western blot analysis of PMCA material (B) shows a higher proportion of RML-infected wild type mice (solid line) positive by round 1 of PMCA, (top blot), whereas most knockout mice (bottom blot, dotted line) required 2 or more rounds of PMCA to visualize PrP Sc . RML (far right) represents 0.05% RML5 amplified prions and serves as a positive control for each PMCA round. ANOVA analysis revealed no significant differences at 30 dpi, but significant differences between the genotypes at 60, 90, and 120 dpi ((p=0.0236, 0.0021, and 0.0032, respectively). Samples in first lane of each blot were not PK digested. Numbers > 100 rpu in the grey area in graph (C) represent scores from samples detected without amplification, and therefore fall outside the dynamic range of PMCA.

Techniques Used: Mouse Assay, Molecular Weight, Infection, Western Blot, Knock-Out, Amplification, Positive Control

28) Product Images from "Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment"

Article Title: Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003355

Assembly of the p7 mutants is impaired prior to capsid envelopment. WT-Jc1- or mutant-transfected detergent-free cell lysates were subjected to a proteolytic digestion protection assay as follows. Lysates were separated into three aliquots which received different treatments: (i) left untreated, (ii) treated with 50 µg/ml proteinase K for 1 h on ice, or (iii) lysed in 5% Triton X-100 prior to proteinase K treatment (condition used for background correction). The amount of protease-resistant core was determined by Western Blot and ELISA. (A) Representative Western Blot stained for HCV core. (B) Western Blot signal intensities were quantified with LabImage 1D and values obtained for the proteinase K-treated sample were background-corrected and normalized to untreated control. Mean values and standard deviations of 3–6 independent experiments are shown.
Figure Legend Snippet: Assembly of the p7 mutants is impaired prior to capsid envelopment. WT-Jc1- or mutant-transfected detergent-free cell lysates were subjected to a proteolytic digestion protection assay as follows. Lysates were separated into three aliquots which received different treatments: (i) left untreated, (ii) treated with 50 µg/ml proteinase K for 1 h on ice, or (iii) lysed in 5% Triton X-100 prior to proteinase K treatment (condition used for background correction). The amount of protease-resistant core was determined by Western Blot and ELISA. (A) Representative Western Blot stained for HCV core. (B) Western Blot signal intensities were quantified with LabImage 1D and values obtained for the proteinase K-treated sample were background-corrected and normalized to untreated control. Mean values and standard deviations of 3–6 independent experiments are shown.

Techniques Used: Mutagenesis, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Characterization of differentially-sedimenting core complexes. Lysates of cells transfected with WT or mutant Jc1 were separated by rate zonal density gradient centrifugation. Each fraction was analyzed for infectivity by TCID 50  (A), core protein content by ELISA (B), and RNA copy number by qRT-PCR (C). Fractions 4 and 7 are highlighted with black arrows. (D) Fractions 4 and 7 were subjected to a proteolytic digestion protection assay and the amount of protease-resistant core protein was quantified by ELISA. The values obtained for the proteinase K-treated samples were background-subtracted and normalized to the untreated controls. Mean values and standard deviations of 2 independent experiments are shown. (E) Lysates of WT Jc1-transfected cells were separated by rate zonal density gradient centrifugation in the absence or presence of 1% DDM and core distribution along the gradient was determined by ELISA.
Figure Legend Snippet: Characterization of differentially-sedimenting core complexes. Lysates of cells transfected with WT or mutant Jc1 were separated by rate zonal density gradient centrifugation. Each fraction was analyzed for infectivity by TCID 50 (A), core protein content by ELISA (B), and RNA copy number by qRT-PCR (C). Fractions 4 and 7 are highlighted with black arrows. (D) Fractions 4 and 7 were subjected to a proteolytic digestion protection assay and the amount of protease-resistant core protein was quantified by ELISA. The values obtained for the proteinase K-treated samples were background-subtracted and normalized to the untreated controls. Mean values and standard deviations of 2 independent experiments are shown. (E) Lysates of WT Jc1-transfected cells were separated by rate zonal density gradient centrifugation in the absence or presence of 1% DDM and core distribution along the gradient was determined by ELISA.

Techniques Used: Transfection, Mutagenesis, Gradient Centrifugation, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Mutations in p7 affect capsid assembly. (A) WT-Jc1 or mutant-transfected cells were harvested 48 h post-transfection. Cell lysates were subjected to rate zonal density gradient centrifugation in the presence of non-ionic detergent (1% DDM) and core protein amounts along the gradient were measured by ELISA. Fractions 3 to 5 are highlighted with black arrows. The right panel shows the core amount relative to total core expression for fraction 5. (B) RNase digestion protection assay (see Materials and Methods ). Fractions 3 to 5 of the WT-Jc1 gradient were subjected to RNase digestion or left untreated. As a background control, samples were treated with proteinase K to remove protecting capsids, prior to RNase treatment. The amount of residual HCV RNA was quantified by qRT-PCR. In parallel crude HCV replication complexes (CRCs) were prepared by the method described by Quinkert et al. [62] . CRCs were then subjected to RNase treatment as described above. To mimic conditions of the sedimentation 1% DDM was added. (C) Separation of core complexes by blue-native-PAGE (4–16% gradient gel). (D) Two-dimensional gel electrophoresis of core complexes. Cell lysates were separated by blue native-PAGE (3–12% gradient gel) in a first electrophoresis and by SDS-PAGE in the second dimension. The results of three independent experiments are shown side by side. Gel pictures are shown vertically for easier comparison with the blue-native-PAGE results. (C, D) Core protein was detected by Western Blotting and subsequent immunodetection with the core-specific monoclonal antibody C7-50.
Figure Legend Snippet: Mutations in p7 affect capsid assembly. (A) WT-Jc1 or mutant-transfected cells were harvested 48 h post-transfection. Cell lysates were subjected to rate zonal density gradient centrifugation in the presence of non-ionic detergent (1% DDM) and core protein amounts along the gradient were measured by ELISA. Fractions 3 to 5 are highlighted with black arrows. The right panel shows the core amount relative to total core expression for fraction 5. (B) RNase digestion protection assay (see Materials and Methods ). Fractions 3 to 5 of the WT-Jc1 gradient were subjected to RNase digestion or left untreated. As a background control, samples were treated with proteinase K to remove protecting capsids, prior to RNase treatment. The amount of residual HCV RNA was quantified by qRT-PCR. In parallel crude HCV replication complexes (CRCs) were prepared by the method described by Quinkert et al. [62] . CRCs were then subjected to RNase treatment as described above. To mimic conditions of the sedimentation 1% DDM was added. (C) Separation of core complexes by blue-native-PAGE (4–16% gradient gel). (D) Two-dimensional gel electrophoresis of core complexes. Cell lysates were separated by blue native-PAGE (3–12% gradient gel) in a first electrophoresis and by SDS-PAGE in the second dimension. The results of three independent experiments are shown side by side. Gel pictures are shown vertically for easier comparison with the blue-native-PAGE results. (C, D) Core protein was detected by Western Blotting and subsequent immunodetection with the core-specific monoclonal antibody C7-50.

Techniques Used: Mutagenesis, Transfection, Gradient Centrifugation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Sedimentation, Blue Native PAGE, Two-Dimensional Gel Electrophoresis, Electrophoresis, SDS Page, Western Blot, Immunodetection

29) Product Images from "Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model"

Article Title: Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model

Journal: Emerging Infectious Diseases

doi: 10.3201/eid13112.070635

Western blot analyses of protease-resistant prion protein from proteinase K–treated brain homogenates from cattle transmissible spongiform encephalopathies (TSEs). Typical bovine spongiform encephalopathy (BSE) (lanes 1, 5), L-type BSE (lane 2), transmissible mink encephalopathy (TME) in cattle (lane 3), H-type BSE (lane 4). Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions.
Figure Legend Snippet: Western blot analyses of protease-resistant prion protein from proteinase K–treated brain homogenates from cattle transmissible spongiform encephalopathies (TSEs). Typical bovine spongiform encephalopathy (BSE) (lanes 1, 5), L-type BSE (lane 2), transmissible mink encephalopathy (TME) in cattle (lane 3), H-type BSE (lane 4). Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions.

Techniques Used: Western Blot, Marker

Western blot of protease-resistant prion protein from TgOvPrP4 mice after proteinase K digestion and immunodetection with anti-PrP Sha31 antibody. A) First passage of typical bovine spongiform encephalopathy (BSE) (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). B) First passage of TME in cattle (lanes 2, 4, and 6) and L-type (lanes 3, 5, and 7). C) Second passage of TME in cattle (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). Each lane shows PrP res from a distinct individual mouse from each experimental group. Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions. Lane 1, PrP res control from a scrapie-infected TgOvPrP4 mouse (C506M3 strain).
Figure Legend Snippet: Western blot of protease-resistant prion protein from TgOvPrP4 mice after proteinase K digestion and immunodetection with anti-PrP Sha31 antibody. A) First passage of typical bovine spongiform encephalopathy (BSE) (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). B) First passage of TME in cattle (lanes 2, 4, and 6) and L-type (lanes 3, 5, and 7). C) Second passage of TME in cattle (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). Each lane shows PrP res from a distinct individual mouse from each experimental group. Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions. Lane 1, PrP res control from a scrapie-infected TgOvPrP4 mouse (C506M3 strain).

Techniques Used: Western Blot, Mouse Assay, Immunodetection, Marker, Infection

30) Product Images from "Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model"

Article Title: Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model

Journal: Emerging Infectious Diseases

doi: 10.3201/eid13112.070635

Western blot analyses of protease-resistant prion protein from proteinase K–treated brain homogenates from cattle transmissible spongiform encephalopathies (TSEs). Typical bovine spongiform encephalopathy (BSE) (lanes 1, 5), L-type BSE (lane 2), transmissible mink encephalopathy (TME) in cattle (lane 3), H-type BSE (lane 4). Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions.
Figure Legend Snippet: Western blot analyses of protease-resistant prion protein from proteinase K–treated brain homogenates from cattle transmissible spongiform encephalopathies (TSEs). Typical bovine spongiform encephalopathy (BSE) (lanes 1, 5), L-type BSE (lane 2), transmissible mink encephalopathy (TME) in cattle (lane 3), H-type BSE (lane 4). Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions.

Techniques Used: Western Blot, Marker

Western blot of protease-resistant prion protein from TgOvPrP4 mice after proteinase K digestion and immunodetection with anti-PrP Sha31 antibody. A) First passage of typical bovine spongiform encephalopathy (BSE) (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). B) First passage of TME in cattle (lanes 2, 4, and 6) and L-type (lanes 3, 5, and 7). C) Second passage of TME in cattle (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). Each lane shows PrP res from a distinct individual mouse from each experimental group. Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions. Lane 1, PrP res control from a scrapie-infected TgOvPrP4 mouse (C506M3 strain).
Figure Legend Snippet: Western blot of protease-resistant prion protein from TgOvPrP4 mice after proteinase K digestion and immunodetection with anti-PrP Sha31 antibody. A) First passage of typical bovine spongiform encephalopathy (BSE) (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). B) First passage of TME in cattle (lanes 2, 4, and 6) and L-type (lanes 3, 5, and 7). C) Second passage of TME in cattle (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). Each lane shows PrP res from a distinct individual mouse from each experimental group. Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions. Lane 1, PrP res control from a scrapie-infected TgOvPrP4 mouse (C506M3 strain).

Techniques Used: Western Blot, Mouse Assay, Immunodetection, Marker, Infection

31) Product Images from "Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype"

Article Title: Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype

Journal: Journal of Virology

doi: 10.1128/JVI.01586-15

Antigenic mapping of PrP Sc from TME and CWD infection of transgenic mice and Syrian golden hamsters. (A) Prion-infected brain homogenates from HPrP7752KO mice and Syrian golden hamsters were digested with or without proteinase K (PK) and analyzed by SDS-PAGE
Figure Legend Snippet: Antigenic mapping of PrP Sc from TME and CWD infection of transgenic mice and Syrian golden hamsters. (A) Prion-infected brain homogenates from HPrP7752KO mice and Syrian golden hamsters were digested with or without proteinase K (PK) and analyzed by SDS-PAGE

Techniques Used: Infection, Transgenic Assay, Mouse Assay, SDS Page

Enrichment and deglycosylation of PrP Sc from TME and CWD strains. Brain homogenates from SGH infected with HY TME, DY TME, WST CWD, and CKY CWD were enriched for PrP Sc by detergent extraction, ultracentrifugation, and proteinase K digestion (A), and N-linked
Figure Legend Snippet: Enrichment and deglycosylation of PrP Sc from TME and CWD strains. Brain homogenates from SGH infected with HY TME, DY TME, WST CWD, and CKY CWD were enriched for PrP Sc by detergent extraction, ultracentrifugation, and proteinase K digestion (A), and N-linked

Techniques Used: Infection

32) Product Images from "Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface"

Article Title: Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191622

Expression and surface-accessibility of V HH -β domain fusions. (A) Cartoon of the structure of the V HH domain fused to the eight β-domain constructs. The disulfide bond that stabilizes the secondary structure and is likely formed in the periplasm is highlighted in blue. (B) Coomassie-stained SDS-PAGE of whole cell lysates (c) and culture supernatants (m) of MC1061 (left panel) or MC1061 degP ::S210A (right panel) cells expressing V HH -β-domain fusions. Indicated are the detectable V HH -β-domain fusions (*). (C) Western blots incubated with α-Myc of whole cell lysates (c) and culture supernatants (m) of MC1061 cells expressing the V HH -β-domain fusions (*). The expected position of a processed V HH -Myc fusion of 18 kDa is indicated on the left. Note that the right panel is derived from a different blot. Compare the results also with Fig 2S that shows that blots of constructs expressed in E . coli strains DHB4 and DHBA yielded a very similar detection pattern. (D) Coomassie-stained SDS-PAGE gels of whole cell lysates of cells of MC1061 cells expressing the V HH -Hbpβ or V HH -IgAPβ(1245) (*) incubated with 100 μg/ml proteinase K (pk) or with buffer (-) for 30 min at 37 °C. The position of the proteinase K bands is indicated by the closed arrowhead, whereas the open arrowheads indicate the control bands used for densitometric analysis ( E) Microscopic images of MC1061 cells expressing V HH -Hbpβ (right panels) or V HH -IgAPβ(1245) (left panels). The top panels are phase-contrast images, the lower panels are immunofluorescent images. Contours of cells are visible due to background autofluoresence.
Figure Legend Snippet: Expression and surface-accessibility of V HH -β domain fusions. (A) Cartoon of the structure of the V HH domain fused to the eight β-domain constructs. The disulfide bond that stabilizes the secondary structure and is likely formed in the periplasm is highlighted in blue. (B) Coomassie-stained SDS-PAGE of whole cell lysates (c) and culture supernatants (m) of MC1061 (left panel) or MC1061 degP ::S210A (right panel) cells expressing V HH -β-domain fusions. Indicated are the detectable V HH -β-domain fusions (*). (C) Western blots incubated with α-Myc of whole cell lysates (c) and culture supernatants (m) of MC1061 cells expressing the V HH -β-domain fusions (*). The expected position of a processed V HH -Myc fusion of 18 kDa is indicated on the left. Note that the right panel is derived from a different blot. Compare the results also with Fig 2S that shows that blots of constructs expressed in E . coli strains DHB4 and DHBA yielded a very similar detection pattern. (D) Coomassie-stained SDS-PAGE gels of whole cell lysates of cells of MC1061 cells expressing the V HH -Hbpβ or V HH -IgAPβ(1245) (*) incubated with 100 μg/ml proteinase K (pk) or with buffer (-) for 30 min at 37 °C. The position of the proteinase K bands is indicated by the closed arrowhead, whereas the open arrowheads indicate the control bands used for densitometric analysis ( E) Microscopic images of MC1061 cells expressing V HH -Hbpβ (right panels) or V HH -IgAPβ(1245) (left panels). The top panels are phase-contrast images, the lower panels are immunofluorescent images. Contours of cells are visible due to background autofluoresence.

Techniques Used: Expressing, Construct, Staining, SDS Page, Western Blot, Incubation, Derivative Assay

Expression and surface-accessibility of Hbp-β domain fusions. (A) Coomassie-stained SDS-PAGE of whole cell lysates (c) and culture supernatants (m) of MC1061 cells expressing wild-type Hbp and Hbp( Spe I). (B) Coomassie-stained gel of whole cell lysates (c) and culture supernatants (m) of MC1061 (left panel) or MC1061 degP ::S210A (right panel) expressing Hbp-β-domain fusions. (C) Coomassie-stained gel of whole cell lysates of cells of MC1061 expressing the Hbp-β-domain fusions incubated with proteinase K, either for 60 min on ice (0°) or 30 min at 37 °C (37°). Included are also untreated controls (-).The positions of bands representing unprocessed Hbp passenger-β-domain fusions (*), and processed Hbp passenger (●) and Hbpβ (▲) are indicated. The latter are only detected for Hbp( Spe I). In panel C, the prominent ~79-kDa of Hbp-Δβcleavage degradation product is indicated by (#), the position of the proteinase K bands is indicated by the closed arrowhead, whereas the open arrowheads indicate the control bands used for densitometric analysis.
Figure Legend Snippet: Expression and surface-accessibility of Hbp-β domain fusions. (A) Coomassie-stained SDS-PAGE of whole cell lysates (c) and culture supernatants (m) of MC1061 cells expressing wild-type Hbp and Hbp( Spe I). (B) Coomassie-stained gel of whole cell lysates (c) and culture supernatants (m) of MC1061 (left panel) or MC1061 degP ::S210A (right panel) expressing Hbp-β-domain fusions. (C) Coomassie-stained gel of whole cell lysates of cells of MC1061 expressing the Hbp-β-domain fusions incubated with proteinase K, either for 60 min on ice (0°) or 30 min at 37 °C (37°). Included are also untreated controls (-).The positions of bands representing unprocessed Hbp passenger-β-domain fusions (*), and processed Hbp passenger (●) and Hbpβ (▲) are indicated. The latter are only detected for Hbp( Spe I). In panel C, the prominent ~79-kDa of Hbp-Δβcleavage degradation product is indicated by (#), the position of the proteinase K bands is indicated by the closed arrowhead, whereas the open arrowheads indicate the control bands used for densitometric analysis.

Techniques Used: Expressing, Staining, SDS Page, Incubation

33) Product Images from "An autotransporter display platform for the development of multivalent recombinant bacterial vector vaccines"

Article Title: An autotransporter display platform for the development of multivalent recombinant bacterial vector vaccines

Journal: Microbial Cell Factories

doi: 10.1186/s12934-014-0162-8

Secretion and display of antigens by attenuated Salmonella Typhimurium. (A-B) Secretion and display of antigens fused to the Hbp passenger. S. Typhimurium SL3261 (unlabeled) and derivatives expressing Hbp-Ag85B [C+N] -ESAT6-Rv2660c or HbpD-Ag85B [C+N] -ESAT6-Rv2660c were analyzed by SDS-PAGE and Coomassie staining (A) or immunoblotting using the indicated antibodies (B) . The equivalent of 0.03 OD 660 units cells (c) and corresponding culture medium (m) samples was analyzed. (C-D) Exposure of antigens at the S. Typhimurium cell surface. (C) SL3261 cells (lane 1–2) and derivatives expressing HbpD-Ag85B [C+N] -ESAT6-Rv2660c from A were treated with Proteinase K (+ pk ) or mock-treated (− pk ). (D) Samples described under C were analyzed by immunoblotting. Cell integrity during the procedure was demonstrated by showing the inaccessibility of the periplasmic chaperone SurA towards Proteinase K using anti-SurA (cf. lanes 1, 3, 5 and 2, 4, 6, resp.). Cleaved Hbp passenger ( > ), non-cleaved Hbp species (*), the cleaved β-domain ( β ) and Proteinase K ( pk ) are indicated. An unrelated protein that cross-reacts with the Hbp β-domain antiserum is indicated ( x ). A proteolytic fragment of HbpD-Ag85B [C+N] -ESAT6-Rv2660c is indicated ( f ). Molecular weight markers (kDa) are shown at the left side of the panels.
Figure Legend Snippet: Secretion and display of antigens by attenuated Salmonella Typhimurium. (A-B) Secretion and display of antigens fused to the Hbp passenger. S. Typhimurium SL3261 (unlabeled) and derivatives expressing Hbp-Ag85B [C+N] -ESAT6-Rv2660c or HbpD-Ag85B [C+N] -ESAT6-Rv2660c were analyzed by SDS-PAGE and Coomassie staining (A) or immunoblotting using the indicated antibodies (B) . The equivalent of 0.03 OD 660 units cells (c) and corresponding culture medium (m) samples was analyzed. (C-D) Exposure of antigens at the S. Typhimurium cell surface. (C) SL3261 cells (lane 1–2) and derivatives expressing HbpD-Ag85B [C+N] -ESAT6-Rv2660c from A were treated with Proteinase K (+ pk ) or mock-treated (− pk ). (D) Samples described under C were analyzed by immunoblotting. Cell integrity during the procedure was demonstrated by showing the inaccessibility of the periplasmic chaperone SurA towards Proteinase K using anti-SurA (cf. lanes 1, 3, 5 and 2, 4, 6, resp.). Cleaved Hbp passenger ( > ), non-cleaved Hbp species (*), the cleaved β-domain ( β ) and Proteinase K ( pk ) are indicated. An unrelated protein that cross-reacts with the Hbp β-domain antiserum is indicated ( x ). A proteolytic fragment of HbpD-Ag85B [C+N] -ESAT6-Rv2660c is indicated ( f ). Molecular weight markers (kDa) are shown at the left side of the panels.

Techniques Used: Expressing, SDS Page, Staining, Molecular Weight

Display of antigenic fragments from C. trachomatis and the influenza A virus by attenuated Salmonella . (A) S . Typhimurium SL3261 cells expressing HbpD(Δd1) or HbpD-MOMP IV -MOMP II . Cells were treated with Proteinase K (+ pk ) or mock-treated (− pk ) before analysis by Coomassie stained SDS-PAGE. (B) Cells from A were analyzed by immunoblotting using antibodies against the Hbp β-domain, chlamydial MOMP or OmpA as indicated. (C) S. Typhimurium SL3261 cells expressing HbpD(Δd1) or HbpD-HA2stem-M2e-NP/PA/M1. Cells were as described under A . (D) Cells from C were analyzed by immunoblotting using antibodies against the Hbp β-domain, influenza M2e and OmpA as indicated. Non-cleaved Hbp species (*), proteolytic fragments of the Hbp-derivatives ( f ) and a truncate of HbpD-HA2stem-M2e-NP/PA/M1 ( > ) are indicated. Molecular weight markers (kDa) are shown at the left side of the panels.
Figure Legend Snippet: Display of antigenic fragments from C. trachomatis and the influenza A virus by attenuated Salmonella . (A) S . Typhimurium SL3261 cells expressing HbpD(Δd1) or HbpD-MOMP IV -MOMP II . Cells were treated with Proteinase K (+ pk ) or mock-treated (− pk ) before analysis by Coomassie stained SDS-PAGE. (B) Cells from A were analyzed by immunoblotting using antibodies against the Hbp β-domain, chlamydial MOMP or OmpA as indicated. (C) S. Typhimurium SL3261 cells expressing HbpD(Δd1) or HbpD-HA2stem-M2e-NP/PA/M1. Cells were as described under A . (D) Cells from C were analyzed by immunoblotting using antibodies against the Hbp β-domain, influenza M2e and OmpA as indicated. Non-cleaved Hbp species (*), proteolytic fragments of the Hbp-derivatives ( f ) and a truncate of HbpD-HA2stem-M2e-NP/PA/M1 ( > ) are indicated. Molecular weight markers (kDa) are shown at the left side of the panels.

Techniques Used: Expressing, Staining, SDS Page, Molecular Weight

34) Product Images from "Accumulation of Pathological Prion Protein PrPSc in the Skin of Animals with Experimental and Natural Scrapie"

Article Title: Accumulation of Pathological Prion Protein PrPSc in the Skin of Animals with Experimental and Natural Scrapie

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0030066

Time-Course of PrP Sc  Deposition in Skin Tissue (A–E) Western blot detection of PrP27–30, the protease-resistant core of PrP Sc , extracted from different skin samples of hamsters orally challenged with 263K scrapie and sacrificed at the following time-points after infection: (A) 70 dpi, (B) 100 dpi, (C) 130 dpi, (D) at the onset of clinical signs for scrapie (138–146 dpi), and (E) at the terminal stage of disease (157–171 dpi). Lanes with test samples: S1, skin sample from hindlimb; S2, skin sample from forelimb; S3, skin sample from back; S4, skin sample from abdomen; S5, skin sample from head. Lanes with control samples: 1, proteinase K–digested brain homogenate from terminally ill 263K scrapie hamsters containing 1 × 10 −7  g brain tissue. Representative results are shown for each stage of incubation. Substantial individual variation was observed at 130 dpi, with two of five and three of five animals displaying findings as in (C) in the Western blot on the left-hand side or the Western blot on the right-hand side, respectively. (F) Lanes S1d–S5d: Same samples as in S1–S5 of (E) but deglycosylated with PNGaseF. (A–F) Amounts of tissue represented in lanes: (A) S1, 43 mg; S2, 52 mg; S3, 68 mg; S4, 58 mg; S5, 73 mg; (B) S1, 78 mg; S2, 44 mg; S3, 63 mg; S4, 67 mg; S5, 50 mg; ([C], Western blot on the left side) S1, 42 mg; S2, 76 mg; S3, 61 mg; S4, 58 mg; S5, 73 mg; ([C], Western blot on the right side) S1, 51 mg; S2, 63 mg; S3, 70 mg; S4, 87 mg; S5, 54 mg; (D) S1, 63 mg; S2, 68 mg; S3, 90 mg; S4, 50 mg; S5, 68 mg; (E) S1, 55 mg; S2, 73 mg; S3, 80 mg; S4, 88 mg; S5, 70 mg; (F) S1d, 12 mg; S2d, 14 mg; S3d, 19 mg; S4d, 12 mg; S5d, 20 mg. (G) Time-scale displaying the mean incubation period and the pre-clinical and clinical phases of incubation of hamsters orally infected with 263K scrapie. Small vertical arrows indicate time-points at which animals were tested for PrP Sc  in skin samples.
Figure Legend Snippet: Time-Course of PrP Sc Deposition in Skin Tissue (A–E) Western blot detection of PrP27–30, the protease-resistant core of PrP Sc , extracted from different skin samples of hamsters orally challenged with 263K scrapie and sacrificed at the following time-points after infection: (A) 70 dpi, (B) 100 dpi, (C) 130 dpi, (D) at the onset of clinical signs for scrapie (138–146 dpi), and (E) at the terminal stage of disease (157–171 dpi). Lanes with test samples: S1, skin sample from hindlimb; S2, skin sample from forelimb; S3, skin sample from back; S4, skin sample from abdomen; S5, skin sample from head. Lanes with control samples: 1, proteinase K–digested brain homogenate from terminally ill 263K scrapie hamsters containing 1 × 10 −7 g brain tissue. Representative results are shown for each stage of incubation. Substantial individual variation was observed at 130 dpi, with two of five and three of five animals displaying findings as in (C) in the Western blot on the left-hand side or the Western blot on the right-hand side, respectively. (F) Lanes S1d–S5d: Same samples as in S1–S5 of (E) but deglycosylated with PNGaseF. (A–F) Amounts of tissue represented in lanes: (A) S1, 43 mg; S2, 52 mg; S3, 68 mg; S4, 58 mg; S5, 73 mg; (B) S1, 78 mg; S2, 44 mg; S3, 63 mg; S4, 67 mg; S5, 50 mg; ([C], Western blot on the left side) S1, 42 mg; S2, 76 mg; S3, 61 mg; S4, 58 mg; S5, 73 mg; ([C], Western blot on the right side) S1, 51 mg; S2, 63 mg; S3, 70 mg; S4, 87 mg; S5, 54 mg; (D) S1, 63 mg; S2, 68 mg; S3, 90 mg; S4, 50 mg; S5, 68 mg; (E) S1, 55 mg; S2, 73 mg; S3, 80 mg; S4, 88 mg; S5, 70 mg; (F) S1d, 12 mg; S2d, 14 mg; S3d, 19 mg; S4d, 12 mg; S5d, 20 mg. (G) Time-scale displaying the mean incubation period and the pre-clinical and clinical phases of incubation of hamsters orally infected with 263K scrapie. Small vertical arrows indicate time-points at which animals were tested for PrP Sc in skin samples.

Techniques Used: Western Blot, Infection, Incubation

PrP Sc  Routing to the Skin and to Components of the Lymphoreticular System of Hamsters Challenged via Different Routes with 263K Scrapie Agent (A) Western blot detection of PrP27–30, the protease-resistant core of PrP Sc , in skin specimens from terminally ill scrapie hamsters. Lanes 1, 2, and 3: skin samples from orally mock-infected control hamsters, spiked before extraction with 1 × 10 −6  g, 5 × 10 −6  g, or 1 × 10 −5  g of brain homogenate from terminally ill 263K hamsters. Lanes 4 and 5: skin samples from hindlimbs and forelimbs of hamsters orally infected with scrapie brain homogenate. Lanes 6 and 7: skin samples from hindlimbs and forelimbs of hamsters intracerebrally infected with scrapie brain homogenate. Lanes 8 and 9: skin samples from hindlimbs and forelimbs of hamsters infected by implantation of s.w. contaminated with scrapie agent. Lanes 10 and 11: skin samples from hindlimbs and forelimbs of hamsters infected peripherally by f.p. inoculation of scrapie brain homogenate. Lanes 12 and 13: skin samples from hindlimbs and forelimbs of hamsters orally mock-infected with normal brain homogenate. Amounts of tissue represented in lanes: 1, 53mg; 2, 58 mg; 3, 68 mg; 4, 68 mg; 5, 75 mg; 6, 78 mg; 7, 64 mg; 8, 69 mg; 9, 60 mg; 10, 62 mg; 11, 73 mg; 12, 61 mg; 13, 58 mg. (B) Western blot detection of PrP27–30 in spleens and selected lymph nodes from terminally ill scrapie hamsters. Lanes 1 and 5: proteinase K-digested brain homogenate from terminally ill scrapie hamsters, containing 1 × 10 −7  g brain tissue. Lanes 2–4: spleen samples from p.o.- (2), s.w.-, (3) and i.c.-infected (4) hamsters. Lanes 6–8: mesenteric lymph node samples from p.o.- (6), s.w.-, (7) and i.c.-infected (8) hamsters. Amounts of tissue represented in lanes: 2, 40 mg; 3, 45 mg; 4, 41 mg; 6, 6 mg; 7, 8 mg; 8, 6 mg.
Figure Legend Snippet: PrP Sc Routing to the Skin and to Components of the Lymphoreticular System of Hamsters Challenged via Different Routes with 263K Scrapie Agent (A) Western blot detection of PrP27–30, the protease-resistant core of PrP Sc , in skin specimens from terminally ill scrapie hamsters. Lanes 1, 2, and 3: skin samples from orally mock-infected control hamsters, spiked before extraction with 1 × 10 −6 g, 5 × 10 −6 g, or 1 × 10 −5 g of brain homogenate from terminally ill 263K hamsters. Lanes 4 and 5: skin samples from hindlimbs and forelimbs of hamsters orally infected with scrapie brain homogenate. Lanes 6 and 7: skin samples from hindlimbs and forelimbs of hamsters intracerebrally infected with scrapie brain homogenate. Lanes 8 and 9: skin samples from hindlimbs and forelimbs of hamsters infected by implantation of s.w. contaminated with scrapie agent. Lanes 10 and 11: skin samples from hindlimbs and forelimbs of hamsters infected peripherally by f.p. inoculation of scrapie brain homogenate. Lanes 12 and 13: skin samples from hindlimbs and forelimbs of hamsters orally mock-infected with normal brain homogenate. Amounts of tissue represented in lanes: 1, 53mg; 2, 58 mg; 3, 68 mg; 4, 68 mg; 5, 75 mg; 6, 78 mg; 7, 64 mg; 8, 69 mg; 9, 60 mg; 10, 62 mg; 11, 73 mg; 12, 61 mg; 13, 58 mg. (B) Western blot detection of PrP27–30 in spleens and selected lymph nodes from terminally ill scrapie hamsters. Lanes 1 and 5: proteinase K-digested brain homogenate from terminally ill scrapie hamsters, containing 1 × 10 −7 g brain tissue. Lanes 2–4: spleen samples from p.o.- (2), s.w.-, (3) and i.c.-infected (4) hamsters. Lanes 6–8: mesenteric lymph node samples from p.o.- (6), s.w.-, (7) and i.c.-infected (8) hamsters. Amounts of tissue represented in lanes: 2, 40 mg; 3, 45 mg; 4, 41 mg; 6, 6 mg; 7, 8 mg; 8, 6 mg.

Techniques Used: Western Blot, Infection

35) Product Images from "UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions"

Article Title: UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions

Journal: Journal of Photochemistry and Photobiology. B, Biology

doi: 10.1016/j.jphotobiol.2015.02.012

Crosslink formation and cell sensitivity. (A) DNA-protein crosslinks: HeLa cells were grown for 48 h in the presence of SIdU or SBrdU as indicated and irradiated with 0, 25 or 50 kJ/m 2 UVA. Cells were lysed immediately after irradiation. Equal amounts of lysate were incubated overnight at 50 °C in the presence (+Prot K) or absence (−Prot K) of Proteinase K. Proteins were then precipitated and discarded. DNA recovered from the supernatant was quantified. Data are expressed as a percentage of recovery from unirradiated cells and are representative of three independent determinations. (B) Crosslink formation – comet assay: HeLa cells grown in the presence of 100 μM SIdU or SBrdU for 48 h were UVA irradiated (20 kJ/m 2 ). They were then γ-irradiated with 15 Gy and analysed by the alkaline comet assay either immediately after irradiation or following Proteinase K digestion as indicated. DNA breakage is expressed as comet tail moment. (C) FANCD2 activation: HeLa cells that had been treated for 24 h with 25 μM SIdU or SBrdU were irradiated with the indicated UVA doses. 2 h after irradiation, cell extracts were analysed by western blotting with FANCD2 antibody. The positions of FANCD2 and its monoubiquitinated form ( ∗ ) are indicated. (D) Sensitivity of Fanconi anemia defective cells: FancD2-proficient (WT) and -deficient (FANCD2) MEFs were grown for 18 h in the presence of 5 μM SIdU (left panel) or 10 μM SBrdU (right panel) to obtain similar levels of DNA substitution. Following UVA-irradiation at the indicated doses, survival was assessed by colony formation. A representative experiment (of three) is shown.
Figure Legend Snippet: Crosslink formation and cell sensitivity. (A) DNA-protein crosslinks: HeLa cells were grown for 48 h in the presence of SIdU or SBrdU as indicated and irradiated with 0, 25 or 50 kJ/m 2 UVA. Cells were lysed immediately after irradiation. Equal amounts of lysate were incubated overnight at 50 °C in the presence (+Prot K) or absence (−Prot K) of Proteinase K. Proteins were then precipitated and discarded. DNA recovered from the supernatant was quantified. Data are expressed as a percentage of recovery from unirradiated cells and are representative of three independent determinations. (B) Crosslink formation – comet assay: HeLa cells grown in the presence of 100 μM SIdU or SBrdU for 48 h were UVA irradiated (20 kJ/m 2 ). They were then γ-irradiated with 15 Gy and analysed by the alkaline comet assay either immediately after irradiation or following Proteinase K digestion as indicated. DNA breakage is expressed as comet tail moment. (C) FANCD2 activation: HeLa cells that had been treated for 24 h with 25 μM SIdU or SBrdU were irradiated with the indicated UVA doses. 2 h after irradiation, cell extracts were analysed by western blotting with FANCD2 antibody. The positions of FANCD2 and its monoubiquitinated form ( ∗ ) are indicated. (D) Sensitivity of Fanconi anemia defective cells: FancD2-proficient (WT) and -deficient (FANCD2) MEFs were grown for 18 h in the presence of 5 μM SIdU (left panel) or 10 μM SBrdU (right panel) to obtain similar levels of DNA substitution. Following UVA-irradiation at the indicated doses, survival was assessed by colony formation. A representative experiment (of three) is shown.

Techniques Used: Irradiation, Incubation, Single Cell Gel Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Activation Assay, Western Blot

Singlet oxygen production and oxidation of DNA. (A) Singlet oxygen and ICL formation: HeLa cells grown in the presence of 200 μM SIdU or SBrdU for 48 h were incubated for 1 h in PBS made in H 2 O or D 2 O and then irradiated with UVA (20 kJ/m 2 ) followed by 15 Gy γ-rays. Analysis was by the alkaline comet assay following Proteinase K digestion. DNA breakage is expressed as comet tail moment. (B) Generation of 8-oxoguanine: HeLa cells were treated with 100 μM SBrdU or SIdU for 48 h and UVA irradiated. The presence of DNA 8-oxoguanine was revealed by single cell electrophoresis following digestion with recombinant OGG1 as indicated. Right panel: In a control experiment HeLa cells were treated with 10 mM KBrO 3 for 1 h and analysed in the same way.
Figure Legend Snippet: Singlet oxygen production and oxidation of DNA. (A) Singlet oxygen and ICL formation: HeLa cells grown in the presence of 200 μM SIdU or SBrdU for 48 h were incubated for 1 h in PBS made in H 2 O or D 2 O and then irradiated with UVA (20 kJ/m 2 ) followed by 15 Gy γ-rays. Analysis was by the alkaline comet assay following Proteinase K digestion. DNA breakage is expressed as comet tail moment. (B) Generation of 8-oxoguanine: HeLa cells were treated with 100 μM SBrdU or SIdU for 48 h and UVA irradiated. The presence of DNA 8-oxoguanine was revealed by single cell electrophoresis following digestion with recombinant OGG1 as indicated. Right panel: In a control experiment HeLa cells were treated with 10 mM KBrO 3 for 1 h and analysed in the same way.

Techniques Used: Incubation, Irradiation, Alkaline Single Cell Gel Electrophoresis, Electrophoresis, Recombinant

36) Product Images from "Prion Shedding from Olfactory Neurons into Nasal Secretions"

Article Title: Prion Shedding from Olfactory Neurons into Nasal Secretions

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000837

Western blot for olfactory marker protein and prion protein in brainstem, olfactory bulb, and olfactory mucosa following HY TME infection of hamsters. Tissue from age-matched, mock-infected (lanes 1, 4, 7) and HY TME-infected (lanes 2, 3, 5, 6, 8, 9) hamsters following intracerebral (lanes 2, 5, 8), and intra-olfactory bulb (lanes 1, 3, 4, 6, 7, 9) inoculation were homogenized and 50 ( A , B ) or 100 ( C ) micrograms of protein was analyzed per lane. In C , protein samples were digested with 10 ug/ml of proteinase K for 37°C for 1 hour prior to SDS-PAGE. This treatment removes PrP C in mock-infected samples and truncates PrP Sc in HY TME lysates. Total prion (PrP) protein ( B ) and PrP Sc ( C ) was immunodetected with anti-PrP 3F4 monoclonal antibody, while olfactory marker protein ( A ) was immunodetected with polyclonal goat serum to OMP. OMP is present in both the olfactory bulb, which contains the olfactory nerve and terminals, and in the olfactory mucosa that contain the soma, dendrites, and axons of ORNs. Marker (M) polypeptides correspond to 20, 30, and 40 kDa. The short vertical tick marks at the top and bottom of each panel are placed before lanes 4 and 7 in order to align lanes between each panel.
Figure Legend Snippet: Western blot for olfactory marker protein and prion protein in brainstem, olfactory bulb, and olfactory mucosa following HY TME infection of hamsters. Tissue from age-matched, mock-infected (lanes 1, 4, 7) and HY TME-infected (lanes 2, 3, 5, 6, 8, 9) hamsters following intracerebral (lanes 2, 5, 8), and intra-olfactory bulb (lanes 1, 3, 4, 6, 7, 9) inoculation were homogenized and 50 ( A , B ) or 100 ( C ) micrograms of protein was analyzed per lane. In C , protein samples were digested with 10 ug/ml of proteinase K for 37°C for 1 hour prior to SDS-PAGE. This treatment removes PrP C in mock-infected samples and truncates PrP Sc in HY TME lysates. Total prion (PrP) protein ( B ) and PrP Sc ( C ) was immunodetected with anti-PrP 3F4 monoclonal antibody, while olfactory marker protein ( A ) was immunodetected with polyclonal goat serum to OMP. OMP is present in both the olfactory bulb, which contains the olfactory nerve and terminals, and in the olfactory mucosa that contain the soma, dendrites, and axons of ORNs. Marker (M) polypeptides correspond to 20, 30, and 40 kDa. The short vertical tick marks at the top and bottom of each panel are placed before lanes 4 and 7 in order to align lanes between each panel.

Techniques Used: Western Blot, Marker, Infection, SDS Page

Western blot for PrP Sc in olfactory bulb and olfactory mucosa following HY TME infection of hamsters. Olfactory bulb (lanes 1 to 4) and olfactory mucosa (lanes 5 to 8) lysates from clinical HY TME hamsters following intracerebral (lanes 1, 2, 5 and 6) and intra-olfactory bulb (lanes 3, 4, 7, and 8) inoculation were enriched for PrP Sc by detergent extraction, ultracentrifugation, and proteinase K digestion. Western blot for prion protein was performed as described in Figure 1 . Marker (M) polypeptides correspond to 20, 30, and 40 kDa.
Figure Legend Snippet: Western blot for PrP Sc in olfactory bulb and olfactory mucosa following HY TME infection of hamsters. Olfactory bulb (lanes 1 to 4) and olfactory mucosa (lanes 5 to 8) lysates from clinical HY TME hamsters following intracerebral (lanes 1, 2, 5 and 6) and intra-olfactory bulb (lanes 3, 4, 7, and 8) inoculation were enriched for PrP Sc by detergent extraction, ultracentrifugation, and proteinase K digestion. Western blot for prion protein was performed as described in Figure 1 . Marker (M) polypeptides correspond to 20, 30, and 40 kDa.

Techniques Used: Western Blot, Infection, Marker

37) Product Images from "Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils"

Article Title: Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Journal: PLoS ONE

doi: 10.1371/journal.pone.0105433

Amyloid fibrils maintained their amyloid architecture after proteolytic digestion and acetone extraction. (A) Aβ 1–40, α-syn or gelsolin peptides (65 µg/ml) in a fibrillar (upper gel) or soluble (lower gel) state were incubated in the absence or presence of 0.13 µg/ml (1∶500, w/w) proteinase K (PK) for 2 h at 42°C. The digestion was conducted in 50 mM sodium phosphate, pH 7.4, 150 mM NaCl buffer. The reaction was stopped by boiling the samples in Laemmli buffer with 2% SDS and the samples were resolved by 16% SDS-PAGE. Western blot using 6E10 (Aβ 1–40 ), syn-1 (α-syn) or a gelsolin-specific antibody is presented. (B) The same reaction described in panel A was performed in the presence of 20 µM of thioflavin T (ThT) and the florescence was monitored every 10 min. Ex = 440 nm and Em = 485 nm. (C) Aβ 1–40 amyloid fibrils at 65 µg/ml concentration were diluted in 1 volume (1 V) of PBS, hexane, acetone or chloroform and centrifuged (16,000 g) for 10 min at 4°C. The pellet was resuspended in phosphate buffer with 20 µM ThT and the fluorescence measured. An aliquot of undiluted/uncentrifuged fibrils was used as the load. Ex = 450 nm and Em = 465–520 nm.
Figure Legend Snippet: Amyloid fibrils maintained their amyloid architecture after proteolytic digestion and acetone extraction. (A) Aβ 1–40, α-syn or gelsolin peptides (65 µg/ml) in a fibrillar (upper gel) or soluble (lower gel) state were incubated in the absence or presence of 0.13 µg/ml (1∶500, w/w) proteinase K (PK) for 2 h at 42°C. The digestion was conducted in 50 mM sodium phosphate, pH 7.4, 150 mM NaCl buffer. The reaction was stopped by boiling the samples in Laemmli buffer with 2% SDS and the samples were resolved by 16% SDS-PAGE. Western blot using 6E10 (Aβ 1–40 ), syn-1 (α-syn) or a gelsolin-specific antibody is presented. (B) The same reaction described in panel A was performed in the presence of 20 µM of thioflavin T (ThT) and the florescence was monitored every 10 min. Ex = 440 nm and Em = 485 nm. (C) Aβ 1–40 amyloid fibrils at 65 µg/ml concentration were diluted in 1 volume (1 V) of PBS, hexane, acetone or chloroform and centrifuged (16,000 g) for 10 min at 4°C. The pellet was resuspended in phosphate buffer with 20 µM ThT and the fluorescence measured. An aliquot of undiluted/uncentrifuged fibrils was used as the load. Ex = 450 nm and Em = 465–520 nm.

Techniques Used: Incubation, SDS Page, Western Blot, Concentration Assay, Fluorescence

Effect of proteinase K digestion and acetone precipitation on the protein content of a complex biological extract. (A and B) The complex biological extract was obtained by mechanical disruption of wild type C. elegans worms followed by a brief centrifugation (700 g for 3 min) to remove unlysed worms. Aβ 1–40 fibrils (0.2% w/w protein concentration) were added to the worm post debris supernatant (PDS) and the samples were digested with PK (1∶500) for 2 h at 42°C followed by acetone precipitation. An aliquot before PK digestion (load), after PK digestion (+ PK) and after PK digestion and acetone precipitation (+ PK/acetone) were resolved by SDS-PAGE (A) or the protein was quantified by BCA assay (B). In the panel A, the upper gel is silver stained and the lower gel is a Western blot for Aβ using the 6E10 antibody. (C–F) TEM images of PDS. PDS was incubated in the absence (C) or in the presence of 0.2% Aβ 1–40 fibrils (E) before the PK/acetone step. PDS incubated in the absence (D) or in the presence of 0.2% Aβ 1–40 fibrils (F) was digested with PK and precipitated with acetone. Note that amyloid fibrils are present only in the samples to which Aβ 1–40 fibrils were added (E and F).
Figure Legend Snippet: Effect of proteinase K digestion and acetone precipitation on the protein content of a complex biological extract. (A and B) The complex biological extract was obtained by mechanical disruption of wild type C. elegans worms followed by a brief centrifugation (700 g for 3 min) to remove unlysed worms. Aβ 1–40 fibrils (0.2% w/w protein concentration) were added to the worm post debris supernatant (PDS) and the samples were digested with PK (1∶500) for 2 h at 42°C followed by acetone precipitation. An aliquot before PK digestion (load), after PK digestion (+ PK) and after PK digestion and acetone precipitation (+ PK/acetone) were resolved by SDS-PAGE (A) or the protein was quantified by BCA assay (B). In the panel A, the upper gel is silver stained and the lower gel is a Western blot for Aβ using the 6E10 antibody. (C–F) TEM images of PDS. PDS was incubated in the absence (C) or in the presence of 0.2% Aβ 1–40 fibrils (E) before the PK/acetone step. PDS incubated in the absence (D) or in the presence of 0.2% Aβ 1–40 fibrils (F) was digested with PK and precipitated with acetone. Note that amyloid fibrils are present only in the samples to which Aβ 1–40 fibrils were added (E and F).

Techniques Used: Centrifugation, Protein Concentration, SDS Page, BIA-KA, Staining, Western Blot, Transmission Electron Microscopy, Incubation

38) Product Images from "Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle"

Article Title: Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00119-15

Effects of PPD administration for skin tests on avidity indices for proteinase K-digested whole-cell sonicate of  M. bovis  (WCS-PK) antigens (A) or MPB83/MPB70 antigens (B) measured by ELISA. Sample collection time points were as follows: post CFT, 14
Figure Legend Snippet: Effects of PPD administration for skin tests on avidity indices for proteinase K-digested whole-cell sonicate of M. bovis (WCS-PK) antigens (A) or MPB83/MPB70 antigens (B) measured by ELISA. Sample collection time points were as follows: post CFT, 14

Techniques Used: Enzyme-linked Immunosorbent Assay

Kinetics of serum antibody responses to complex (WCS-PK) and specific (MPB83/MPB70) antigens and effects of skin tests, as measured by ELISA. (A) ELISA results for proteinase K-digested whole-cell sonicate (WCS-PK) of  M. bovis  over time after an  M. bovis
Figure Legend Snippet: Kinetics of serum antibody responses to complex (WCS-PK) and specific (MPB83/MPB70) antigens and effects of skin tests, as measured by ELISA. (A) ELISA results for proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis over time after an M. bovis

Techniques Used: Enzyme-linked Immunosorbent Assay

39) Product Images from "A structurally informed autotransporter platform for efficient heterologous protein secretion and display"

Article Title: A structurally informed autotransporter platform for efficient heterologous protein secretion and display

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-11-85

Cell surface exposure of HbpD-ESAT6 fusions. ( A-B ) Expression of Hbp display constructs analyzed by Coomassie staining (A) or immunoblotting (B) as described in the legend to Figure 1 . ( C ) Proteinase K accessibility of HbpD-ESAT6 fusions at the cell surface. Part of the cells described under A was collected and resuspended in 50 mM Tris–HCl, pH 7.4, 1 mM CaCl. For Hbp(Δβ-cleav) and HbpD(Δd1), half of the cell suspension was lysed by sonication ( son ) on ice using a tip sonicator (Branson Sonifier 250). Subsequently, all samples were incubated with Proteinase K ( pk ; 100 μg/ml) at 37°C for 1 h. The reaction was stopped by addition of 0.1 mM phenylmethanesulfonylfluoride (PMSF) and incubation on ice for 5 min. Samples were TCA precipitated before analysis by SDS-PAGE and Coomassie staining. Non-cleaved Hbp species (*) are indicated. Molecular mass (kDa) markers are indicated at the left side of the panels.
Figure Legend Snippet: Cell surface exposure of HbpD-ESAT6 fusions. ( A-B ) Expression of Hbp display constructs analyzed by Coomassie staining (A) or immunoblotting (B) as described in the legend to Figure 1 . ( C ) Proteinase K accessibility of HbpD-ESAT6 fusions at the cell surface. Part of the cells described under A was collected and resuspended in 50 mM Tris–HCl, pH 7.4, 1 mM CaCl. For Hbp(Δβ-cleav) and HbpD(Δd1), half of the cell suspension was lysed by sonication ( son ) on ice using a tip sonicator (Branson Sonifier 250). Subsequently, all samples were incubated with Proteinase K ( pk ; 100 μg/ml) at 37°C for 1 h. The reaction was stopped by addition of 0.1 mM phenylmethanesulfonylfluoride (PMSF) and incubation on ice for 5 min. Samples were TCA precipitated before analysis by SDS-PAGE and Coomassie staining. Non-cleaved Hbp species (*) are indicated. Molecular mass (kDa) markers are indicated at the left side of the panels.

Techniques Used: Expressing, Construct, Staining, Sonication, Incubation, SDS Page

Display of ESAT6 by attenuated Salmonella typhimurium. ( A - B ) Expression of HbpD(Δd1)-ESAT6. S. typhimurium SL3261 (-) and a derivative expressing HbpD(Δd1)-ESAT6 were grown to mid-log phase in LB medium at 37°C. The equivalent of 0.03 OD 660 units cells (c) and corresponding culture medium (m) samples was analyzed by SDS-PAGE and Coomassie staining (A) or immunoblotting (B). ( C - E ) Exposure of HbpD(Δd1)-ESAT6 at the S. typhimurium cell surface. ( C ) Cells from A were collected and resuspended in icecold reaction buffer (50 mM Tris HCl, pH 7.4, 1 mM CaCl 2 ). The samples were treated with 100 μg/ml Proteinase K ( + pk ) or mock-treated ( - pk ) at 37°C for 1 h. The reaction was stopped by incubation with PMSF (0.1 mM) for 10 min on ice. Samples were TCA precipitated and analyzed as described under A . ( D - E ) Samples described under C were analyzed by immunoblotting. Cell integrity during the procedure was demonstrated by showing the inaccessibility of the periplasmic chaperone SurA towards Proteinase K using anti-SurA (cf. lanes 1, 3, 5 and 2, 4, 6, resp.). Non-cleaved Hbp species (*) are indicated. Molecular mass (kDa) markers are shown at the left side of the panels.
Figure Legend Snippet: Display of ESAT6 by attenuated Salmonella typhimurium. ( A - B ) Expression of HbpD(Δd1)-ESAT6. S. typhimurium SL3261 (-) and a derivative expressing HbpD(Δd1)-ESAT6 were grown to mid-log phase in LB medium at 37°C. The equivalent of 0.03 OD 660 units cells (c) and corresponding culture medium (m) samples was analyzed by SDS-PAGE and Coomassie staining (A) or immunoblotting (B). ( C - E ) Exposure of HbpD(Δd1)-ESAT6 at the S. typhimurium cell surface. ( C ) Cells from A were collected and resuspended in icecold reaction buffer (50 mM Tris HCl, pH 7.4, 1 mM CaCl 2 ). The samples were treated with 100 μg/ml Proteinase K ( + pk ) or mock-treated ( - pk ) at 37°C for 1 h. The reaction was stopped by incubation with PMSF (0.1 mM) for 10 min on ice. Samples were TCA precipitated and analyzed as described under A . ( D - E ) Samples described under C were analyzed by immunoblotting. Cell integrity during the procedure was demonstrated by showing the inaccessibility of the periplasmic chaperone SurA towards Proteinase K using anti-SurA (cf. lanes 1, 3, 5 and 2, 4, 6, resp.). Non-cleaved Hbp species (*) are indicated. Molecular mass (kDa) markers are shown at the left side of the panels.

Techniques Used: Expressing, SDS Page, Staining, Incubation

40) Product Images from "Generation of a Persistently Infected MDBK Cell Line with Natural Bovine Spongiform Encephalopathy (BSE)"

Article Title: Generation of a Persistently Infected MDBK Cell Line with Natural Bovine Spongiform Encephalopathy (BSE)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115939

Confirmation and cloning of PrP BSE -infected cells. The presence of proteinase K resistant PrP BSE in 96 well elispot plates was confirmed by using scrapie cell assay (SCA). The photograph showed severe (ID5, IIIG1, IVB2, IVF12, VD7 and VG3), moderate (IVC4), weak (IIID9) and non-infected (IF3) wells. These data shown are representative of results from ELISA and Western blot.
Figure Legend Snippet: Confirmation and cloning of PrP BSE -infected cells. The presence of proteinase K resistant PrP BSE in 96 well elispot plates was confirmed by using scrapie cell assay (SCA). The photograph showed severe (ID5, IIIG1, IVB2, IVF12, VD7 and VG3), moderate (IVC4), weak (IIID9) and non-infected (IF3) wells. These data shown are representative of results from ELISA and Western blot.

Techniques Used: Clone Assay, Infection, Enzyme-linked Immunospot, Scrapie Cell Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Immunoblotting detection of the persistent PrP BSE -infected cell according to serial passages. The lysates of brain homogenates (lane 1; BSE-noninfected bovine brain, lane 2; BSE-infected bovine brain-original BSE source) and MDBK cells expressing normal bovine prion protein by using infectious recombinant lentivirus (lane 3: transduced MDBK–MBDK C1–2F–passage(p) 7; lane 4: BSE-infected transduced MDBK (M2B)–p17; lane 5: M2B-p48; lane 6: M2B-p70 and lane 7: M2B-p83) were treated with proteinase K (PK) for detection of PrP BSE in transduced MDBK which was sequentially passaged after inoculating BSE-infected bovine brain homogenate. Molecular mass marker (M) in kilodaltons (kDa) is shown on the left. The result shown is representative of multiple independent experiments.
Figure Legend Snippet: Immunoblotting detection of the persistent PrP BSE -infected cell according to serial passages. The lysates of brain homogenates (lane 1; BSE-noninfected bovine brain, lane 2; BSE-infected bovine brain-original BSE source) and MDBK cells expressing normal bovine prion protein by using infectious recombinant lentivirus (lane 3: transduced MDBK–MBDK C1–2F–passage(p) 7; lane 4: BSE-infected transduced MDBK (M2B)–p17; lane 5: M2B-p48; lane 6: M2B-p70 and lane 7: M2B-p83) were treated with proteinase K (PK) for detection of PrP BSE in transduced MDBK which was sequentially passaged after inoculating BSE-infected bovine brain homogenate. Molecular mass marker (M) in kilodaltons (kDa) is shown on the left. The result shown is representative of multiple independent experiments.

Techniques Used: Infection, Expressing, Recombinant, Marker

41) Product Images from "Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc"

Article Title: Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

Journal: PeerJ

doi: 10.7717/peerj.811

N-terminal mAbs can inhibit prion replication. RML infected GT1 cells were treated for 6 days with increasing concentrations (0, 1, 2.5, 5 and 7.5 µg/mL) of EB8, DE10, DC2 and EF2 mAbs, refreshing medium the third day. Cell lysates were digested with proteinase K (PK + lanes) and PrP Sc levels checked by Western blot using Fab D18 for detection. As positive control (PC), cell lysates from uninfected GT1 cells were also digested. About 25 µg of total proteins were loaded as control (PK—lanes). DE10, DC2 and EF2 mAbs promoted a complete clearance of prions starting from the lowest concentration tested whilst cells treated with EB8 showed a residual signal of PrP Sc even at the highest concentration of antibody. Images are representative of three independent experiments.
Figure Legend Snippet: N-terminal mAbs can inhibit prion replication. RML infected GT1 cells were treated for 6 days with increasing concentrations (0, 1, 2.5, 5 and 7.5 µg/mL) of EB8, DE10, DC2 and EF2 mAbs, refreshing medium the third day. Cell lysates were digested with proteinase K (PK + lanes) and PrP Sc levels checked by Western blot using Fab D18 for detection. As positive control (PC), cell lysates from uninfected GT1 cells were also digested. About 25 µg of total proteins were loaded as control (PK—lanes). DE10, DC2 and EF2 mAbs promoted a complete clearance of prions starting from the lowest concentration tested whilst cells treated with EB8 showed a residual signal of PrP Sc even at the highest concentration of antibody. Images are representative of three independent experiments.

Techniques Used: Infection, Western Blot, Positive Control, Concentration Assay

Time-course analysis of mAb-induced prion clearance. ScGT1 cells were incubated for 1 week with 5 µg/mL of mAbs. Untreated cells were used as negative control (NC). After the initial treatment, cells were split and cultured in absence of mAbs for 1 month. Cell lysates were digested with proteinase K (PK + lanes) and PrP Sc was probed by Western blot using Fab D18. PrP Sc levels were analyzed after one (1 w), two (2 w), three (3 w) and four (4 w) weeks after the treatment to evaluate the stability of clearance during time. Prions were not detectable in treated cells one month after the mAbs incubation. Just a slight signal from PrP Sc was found in EB8 treated ScGT1 cells. Images are representative of three independent experiments. Lanes were run on the same gel but were non-contiguous (white lines).
Figure Legend Snippet: Time-course analysis of mAb-induced prion clearance. ScGT1 cells were incubated for 1 week with 5 µg/mL of mAbs. Untreated cells were used as negative control (NC). After the initial treatment, cells were split and cultured in absence of mAbs for 1 month. Cell lysates were digested with proteinase K (PK + lanes) and PrP Sc was probed by Western blot using Fab D18. PrP Sc levels were analyzed after one (1 w), two (2 w), three (3 w) and four (4 w) weeks after the treatment to evaluate the stability of clearance during time. Prions were not detectable in treated cells one month after the mAbs incubation. Just a slight signal from PrP Sc was found in EB8 treated ScGT1 cells. Images are representative of three independent experiments. Lanes were run on the same gel but were non-contiguous (white lines).

Techniques Used: Incubation, Negative Control, Cell Culture, Western Blot

42) Product Images from "Environmentally-Relevant Forms of the Prion Protein"

Article Title: Environmentally-Relevant Forms of the Prion Protein

Journal:

doi:

HY TME Degradation. On the left: representative immunoblots of 2.5 µL of 10% BH (pH 4, 7, or 10) after incubation for up to 35 days. Samples were not treated with proteinase K. On the right: signal intensities of sample blots ( n  = 4 or 5) were
Figure Legend Snippet: HY TME Degradation. On the left: representative immunoblots of 2.5 µL of 10% BH (pH 4, 7, or 10) after incubation for up to 35 days. Samples were not treated with proteinase K. On the right: signal intensities of sample blots ( n = 4 or 5) were

Techniques Used: Western Blot, Incubation

PrP Sc Detection Using Various Anti-PrP Antibodies. Panel A: Representative immunoblots of HY TME agent-infected brain homogenate using different antibodies, with (+) or without (−) proteinase K (PK) treatment. Panel B: A schematic representation
Figure Legend Snippet: PrP Sc Detection Using Various Anti-PrP Antibodies. Panel A: Representative immunoblots of HY TME agent-infected brain homogenate using different antibodies, with (+) or without (−) proteinase K (PK) treatment. Panel B: A schematic representation

Techniques Used: Western Blot, Infection

CWD Degradation. On the left: representative immunoblots of 2.5 µL of 10% BH (pH 7) after incubation for up to 35 days. Samples were not treated with proteinase K. On the right: signal intensities of sample blots ( n  = 4 or 5) were normalized against
Figure Legend Snippet: CWD Degradation. On the left: representative immunoblots of 2.5 µL of 10% BH (pH 7) after incubation for up to 35 days. Samples were not treated with proteinase K. On the right: signal intensities of sample blots ( n = 4 or 5) were normalized against

Techniques Used: Western Blot, Incubation

43) Product Images from "In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae"

Article Title: In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18040702

Revised topology model of Chs3 based on the combined results from previous interaction studies and our bioinformatic and biochemical analysis. C-myc insertions for proteinase K experiments are marked by black triangles, and the α-Chs3 antibody binding site is indicated by an open grey triangle. Conserved motifs are marked by grey circles or diamonds. Phosphorylation sites are indicated by P (diamonds), N -glycosylation sites by N (circle), the ubiquitination site by u (circle), and the palmitoylation site by P (circle).
Figure Legend Snippet: Revised topology model of Chs3 based on the combined results from previous interaction studies and our bioinformatic and biochemical analysis. C-myc insertions for proteinase K experiments are marked by black triangles, and the α-Chs3 antibody binding site is indicated by an open grey triangle. Conserved motifs are marked by grey circles or diamonds. Phosphorylation sites are indicated by P (diamonds), N -glycosylation sites by N (circle), the ubiquitination site by u (circle), and the palmitoylation site by P (circle).

Techniques Used: Binding Assay

Topology determination of Chs3 using Proteinase K experiments with different myc-epitope tagged versions of Chs3. Cells expressing Chs3 13myc or different Chs3 3myc versions were converted into spheroplasts and treated with or without proteinase K in the presence or absence of Triton X-100. TCA precipitated proteins were solubilized and analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and Western blots using either: α-Chs3 or α-myc ( A ); and α-phosphofructokinase (PFK) ( B ) for immunostaining. Detection of PFK serves as a control for the integrity of the spheroplasts. Standard proteins are indicated with molecular masses given in kDa.
Figure Legend Snippet: Topology determination of Chs3 using Proteinase K experiments with different myc-epitope tagged versions of Chs3. Cells expressing Chs3 13myc or different Chs3 3myc versions were converted into spheroplasts and treated with or without proteinase K in the presence or absence of Triton X-100. TCA precipitated proteins were solubilized and analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and Western blots using either: α-Chs3 or α-myc ( A ); and α-phosphofructokinase (PFK) ( B ) for immunostaining. Detection of PFK serves as a control for the integrity of the spheroplasts. Standard proteins are indicated with molecular masses given in kDa.

Techniques Used: Expressing, Electrophoresis, SDS Page, Western Blot, Immunostaining

44) Product Images from "Structural properties of the tubular appendage spinae from marine bacterium Roseobacter sp. strain YSCB"

Article Title: Structural properties of the tubular appendage spinae from marine bacterium Roseobacter sp. strain YSCB

Journal: Scientific Reports

doi: 10.1038/srep00950

Disintegration of Roseobacter sp. YSCB spinae upon proteolysis or thermal treatments. Purified spinae were examined with SDS-PAGE on 12.5% gels (A, sizes of protein markers on left) or by TEM (negatively stained) without treatment (A, lane 1 and B), or digested with proteinase K at a concentration of 2 mg/ml (A, lane 2; C1), 10 mg/ml (A, lane 3; C2) and 20 mg/ml (A, lane 4; C3), or heated at 100°C for 1 min (D1), 5 min (D2), 10 min (D3 and D4). Insert in C2 shows a single filament winding into the tubular structure. The arrow in B indicates flagellar filament. Asterisks in panel A indicate proteolysis products.
Figure Legend Snippet: Disintegration of Roseobacter sp. YSCB spinae upon proteolysis or thermal treatments. Purified spinae were examined with SDS-PAGE on 12.5% gels (A, sizes of protein markers on left) or by TEM (negatively stained) without treatment (A, lane 1 and B), or digested with proteinase K at a concentration of 2 mg/ml (A, lane 2; C1), 10 mg/ml (A, lane 3; C2) and 20 mg/ml (A, lane 4; C3), or heated at 100°C for 1 min (D1), 5 min (D2), 10 min (D3 and D4). Insert in C2 shows a single filament winding into the tubular structure. The arrow in B indicates flagellar filament. Asterisks in panel A indicate proteolysis products.

Techniques Used: Purification, SDS Page, Transmission Electron Microscopy, Staining, Concentration Assay

Biophysical characterization of Roseobacter sp. YSCB spinae. Panel A1 (TEM) shows the sample from where the XEDS spectra (A2) were obtained. The peaks correspond to carbon (1), nitrogen (2), oxygen (3), sodium (4), magnesium (5), silicium (6), sulfur (7), chore (8) and calcium (9). Panel B1 is a representative DSC spectrum of the extracted spinae samples. The scan rate is 1°C per min. The thermograms of the subsequent first, second and sixth scan are presented in black (line), red (dash-line) and blue (dot-line), respectively. Panel B2 shows the CD spectroscopy results of the sample before (black line) or after (red dash-line) the first DSC scan. Panel C is IR spectra of amide I bands of the spinae extraction without treatment (black line), or treated at 100°C for 20 min (green dash-line) or with 2 mg/ml proteinase K for overnight at room temperature (blue dot-line). The spectrum of the recombined proteinase K (100 mg/ml) was measured and presented as a reference (red dash-dot-line). The curves are normalized to fit into the same scale.
Figure Legend Snippet: Biophysical characterization of Roseobacter sp. YSCB spinae. Panel A1 (TEM) shows the sample from where the XEDS spectra (A2) were obtained. The peaks correspond to carbon (1), nitrogen (2), oxygen (3), sodium (4), magnesium (5), silicium (6), sulfur (7), chore (8) and calcium (9). Panel B1 is a representative DSC spectrum of the extracted spinae samples. The scan rate is 1°C per min. The thermograms of the subsequent first, second and sixth scan are presented in black (line), red (dash-line) and blue (dot-line), respectively. Panel B2 shows the CD spectroscopy results of the sample before (black line) or after (red dash-line) the first DSC scan. Panel C is IR spectra of amide I bands of the spinae extraction without treatment (black line), or treated at 100°C for 20 min (green dash-line) or with 2 mg/ml proteinase K for overnight at room temperature (blue dot-line). The spectrum of the recombined proteinase K (100 mg/ml) was measured and presented as a reference (red dash-dot-line). The curves are normalized to fit into the same scale.

Techniques Used: Transmission Electron Microscopy, Spectroscopy

45) Product Images from "Transmissible Gastroenteritis Coronavirus RNA-Dependent RNA Polymerase and Nonstructural Proteins 2, 3, and 8 Are Incorporated into Viral Particles"

Article Title: Transmissible Gastroenteritis Coronavirus RNA-Dependent RNA Polymerase and Nonstructural Proteins 2, 3, and 8 Are Incorporated into Viral Particles

Journal: Journal of Virology

doi: 10.1128/JVI.06428-11

Identification of nsps 2, 3, and 8 in TGEV viral particles. (A) Western blot analysis. Proteins from purified TGEV (V) treated (+PK) or not treated (−PK) with proteinase K were separated by SDS-PAGE (4 to 12%) together with extracts from ST cells
Figure Legend Snippet: Identification of nsps 2, 3, and 8 in TGEV viral particles. (A) Western blot analysis. Proteins from purified TGEV (V) treated (+PK) or not treated (−PK) with proteinase K were separated by SDS-PAGE (4 to 12%) together with extracts from ST cells

Techniques Used: Western Blot, Purification, SDS Page

Identification of RdRp in TGEV particles. (A) Analysis of RdRp in viral particles treated with proteinase K. Ten micrograms of highly purified TGEV was not treated (−PK) or treated (+PK) with 0.1 μg of proteinase K, and the presence of
Figure Legend Snippet: Identification of RdRp in TGEV particles. (A) Analysis of RdRp in viral particles treated with proteinase K. Ten micrograms of highly purified TGEV was not treated (−PK) or treated (+PK) with 0.1 μg of proteinase K, and the presence of

Techniques Used: Purification

46) Product Images from "Characterization of Two New Structural Glycoproteins, GP3 and GP4, of Equine Arteritis Virus"

Article Title: Characterization of Two New Structural Glycoproteins, GP3 and GP4, of Equine Arteritis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.76.21.10829-10840.2002

Membrane topology of the GP 3 protein. ORF3 RNA transcripts were translated in vitro in the presence of canine pancreatic microsomes. Subsequently, the translation products were treated or mock treated with proteinase K (prot. K) in the absence of detergent or after disruption of the microsomal membranes with TX-100. After a 1-h incubation at 4°C, the proteinase K was inactivated by protease inhibitors. Next, the samples were immunoprecipitated with a GP 3 -specific antipeptide serum (GP 3 ) or its preimmune serum (pGP 3 ). The resulting immune complexes were each split into two equal portions that were treated or mock treated with PNGase F. The samples were dissolved in LSB containing 5% β-mercaptoethanol and analyzed in SDS-15% PAA gels. The values on the left are the molecular sizes, in kilodaltons, of marker proteins analyzed in the same gel. Ab, antibody.
Figure Legend Snippet: Membrane topology of the GP 3 protein. ORF3 RNA transcripts were translated in vitro in the presence of canine pancreatic microsomes. Subsequently, the translation products were treated or mock treated with proteinase K (prot. K) in the absence of detergent or after disruption of the microsomal membranes with TX-100. After a 1-h incubation at 4°C, the proteinase K was inactivated by protease inhibitors. Next, the samples were immunoprecipitated with a GP 3 -specific antipeptide serum (GP 3 ) or its preimmune serum (pGP 3 ). The resulting immune complexes were each split into two equal portions that were treated or mock treated with PNGase F. The samples were dissolved in LSB containing 5% β-mercaptoethanol and analyzed in SDS-15% PAA gels. The values on the left are the molecular sizes, in kilodaltons, of marker proteins analyzed in the same gel. Ab, antibody.

Techniques Used: In Vitro, Incubation, Immunoprecipitation, Marker

Membrane topology of the GP 4 protein. ORF4 RNA transcripts were translated in vitro in the presence of canine pancreatic microsomes. Subsequently, the samples were treated or mock treated with proteinase K (prot. K) in the absence of a detergent or after disruption of the microsomal membranes with TX-100. After a 1-h incubation at 4°C, the proteinase K was inactivated by addition of protease inhibitors. Next, the samples were split into three equal portions. One aliquot was subjected to IP with a GP 4 -specific antipeptide serum (GP 4 E ), the second aliquot was mixed with the corresponding preimmune serum (pGP 4 E ), and the third aliquot was subjected to IP with αGP 4 E and treated with endo H. The samples were dissolved in LSB containing 5% β-mercaptoethanol and analyzed in SDS-15% PAA gels. The values on the left are the molecular sizes, in kilodaltons, of marker proteins analyzed in the same gel. Ab, antibody.
Figure Legend Snippet: Membrane topology of the GP 4 protein. ORF4 RNA transcripts were translated in vitro in the presence of canine pancreatic microsomes. Subsequently, the samples were treated or mock treated with proteinase K (prot. K) in the absence of a detergent or after disruption of the microsomal membranes with TX-100. After a 1-h incubation at 4°C, the proteinase K was inactivated by addition of protease inhibitors. Next, the samples were split into three equal portions. One aliquot was subjected to IP with a GP 4 -specific antipeptide serum (GP 4 E ), the second aliquot was mixed with the corresponding preimmune serum (pGP 4 E ), and the third aliquot was subjected to IP with αGP 4 E and treated with endo H. The samples were dissolved in LSB containing 5% β-mercaptoethanol and analyzed in SDS-15% PAA gels. The values on the left are the molecular sizes, in kilodaltons, of marker proteins analyzed in the same gel. Ab, antibody.

Techniques Used: In Vitro, Incubation, Marker

47) Product Images from "UK Iatrogenic Creutzfeldt–Jakob disease: investigating human prion transmission across genotypic barriers using human tissue-based and molecular approaches"

Article Title: UK Iatrogenic Creutzfeldt–Jakob disease: investigating human prion transmission across genotypic barriers using human tissue-based and molecular approaches

Journal: Acta Neuropathologica

doi: 10.1007/s00401-016-1638-x

PrP res type found in the iCJD cases examined. a Four PrP res molecular types were detected by Western blot analysis of proteinase K treated brain extracts of hGH-iCJD patients; type 1, type i, type i + 2 and type 2. Representative blots of each PrP res type are shown. Each sample ( middle lane ) is flanked by type 1 (T1) ( left lane ) and type 2 (T2) ( right lane ) reference standards from sCJD MM1 and VV2 subtype cases, respectively. Case number and codon 129 PRNP genotype are indicated above each blot, whilst PrP res type is indicated below. The most informative exposure for the test and reference standard samples from a series of timed exposures is shown. Blots were probed using the monoclonal antibody 3F4. b Western blot analysis of proteinase K treated samples of PrP res types detected in hGH-iCJD patients using the monoclonal antibody 12B2 which detects type 1 PrP res and shown after a prolonged (30 min) and abbreviated (3 min) exposure time. Approximate molecular mass is shown in kDa. The immunoblots shown are representative of at least three technical repeats
Figure Legend Snippet: PrP res type found in the iCJD cases examined. a Four PrP res molecular types were detected by Western blot analysis of proteinase K treated brain extracts of hGH-iCJD patients; type 1, type i, type i + 2 and type 2. Representative blots of each PrP res type are shown. Each sample ( middle lane ) is flanked by type 1 (T1) ( left lane ) and type 2 (T2) ( right lane ) reference standards from sCJD MM1 and VV2 subtype cases, respectively. Case number and codon 129 PRNP genotype are indicated above each blot, whilst PrP res type is indicated below. The most informative exposure for the test and reference standard samples from a series of timed exposures is shown. Blots were probed using the monoclonal antibody 3F4. b Western blot analysis of proteinase K treated samples of PrP res types detected in hGH-iCJD patients using the monoclonal antibody 12B2 which detects type 1 PrP res and shown after a prolonged (30 min) and abbreviated (3 min) exposure time. Approximate molecular mass is shown in kDa. The immunoblots shown are representative of at least three technical repeats

Techniques Used: Western Blot

48) Product Images from "Distinct molecular phenotypes in bovine prion diseases"

Article Title: Distinct molecular phenotypes in bovine prion diseases

Journal: EMBO Reports

doi: 10.1038/sj.embor.7400054

( A ) Western blot detection of PrP res  from proteinase K-treated and ultracentrifuged brain homogenates, using RB1 polyclonal antibody (105–120 bovine epitope). Atypical cattle-BSE cases (A1 F  and A2 F ) (lanes 3 and 5) show, as do scrapie experimentally infected cattle (C Scr ) (lane 1), a higher molecular mass of the unglycosylated bands (arrow), compared to typical cattle-BSE cases (T UK , lane 2; T1 F , lane 4). Similar differences are also observed in sheep, between natural scrapie (SH NS ) (lane 7) and experimental BSE (SH BSE ) (lane 6). Brain equivalent quantities loaded per lane from lanes 1 to 7 are 2.4, 4.8, 38, 2.4, 38, 1.2 and 1.2 mg, respectively.  (B)  Mean molecular weights±standard deviations obtained for the di-, mono- and unglycosylated PrP res  bands (A: atypical cattle BSE; T: typical cattle BSE; F: French isolate; UK: British isolate; SH: sheep).
Figure Legend Snippet: ( A ) Western blot detection of PrP res from proteinase K-treated and ultracentrifuged brain homogenates, using RB1 polyclonal antibody (105–120 bovine epitope). Atypical cattle-BSE cases (A1 F and A2 F ) (lanes 3 and 5) show, as do scrapie experimentally infected cattle (C Scr ) (lane 1), a higher molecular mass of the unglycosylated bands (arrow), compared to typical cattle-BSE cases (T UK , lane 2; T1 F , lane 4). Similar differences are also observed in sheep, between natural scrapie (SH NS ) (lane 7) and experimental BSE (SH BSE ) (lane 6). Brain equivalent quantities loaded per lane from lanes 1 to 7 are 2.4, 4.8, 38, 2.4, 38, 1.2 and 1.2 mg, respectively. (B) Mean molecular weights±standard deviations obtained for the di-, mono- and unglycosylated PrP res bands (A: atypical cattle BSE; T: typical cattle BSE; F: French isolate; UK: British isolate; SH: sheep).

Techniques Used: Western Blot, Infection

49) Product Images from "DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay"

Article Title: DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

Journal: Molecules and Cells

doi: 10.14348/molcells.2016.2206

Analysis of Comet assay. Worms were irradiated with 30 Gy of IR and postincubated for various recovery times. Comet slides were prepared, lysed, treated with glyoxal, and electrophoresed in a neutral buffer. (A) Comet images were captured using an epifluorescence microscope (Carl Zeiss), Scale bar, 50 μm. (B) The tail moment of comets in (A) was analyzed using Comet Assay IV software. Comet slides were prepared, lysed, treated with RNase and proteinase K, and electrophoresed in a neutral buffer. (C) Comet images were captured using an epifluorescence microscope (Carl Zeiss), Scale bar, 50 μm. (D) The tail moment of comets in (C) was analyzed using Comet Assay IV software.
Figure Legend Snippet: Analysis of Comet assay. Worms were irradiated with 30 Gy of IR and postincubated for various recovery times. Comet slides were prepared, lysed, treated with glyoxal, and electrophoresed in a neutral buffer. (A) Comet images were captured using an epifluorescence microscope (Carl Zeiss), Scale bar, 50 μm. (B) The tail moment of comets in (A) was analyzed using Comet Assay IV software. Comet slides were prepared, lysed, treated with RNase and proteinase K, and electrophoresed in a neutral buffer. (C) Comet images were captured using an epifluorescence microscope (Carl Zeiss), Scale bar, 50 μm. (D) The tail moment of comets in (C) was analyzed using Comet Assay IV software.

Techniques Used: Single Cell Gel Electrophoresis, Irradiation, Microscopy, Software

Analysis of Comet assay. Worms were treated with 5 μM CPT for 24 h and postincubated on new fresh NGM plates for various recovery times. Comet slides were prepared, lysed, treated with glyoxal, and electrophoresed in a neutral buffer. (A) Comet images were captured using an epifluorescence microscope (Carl Zeiss), Scale bar, 50 μm. (B) The tail moment of comets in (A) was analyzed using Comet Assay IV software. Comet slides were prepared, lysed, treated with RNase and proteinase K, and electrophoresed in a neutral buffer. (C) Comet images were captured using an epifluorescence microscope (Carl Zeiss), Scale bar, 50 μm. (D) The tail moment of comets in (C) was analyzed using Comet Assay IV software. no, no CPT-treated worms.
Figure Legend Snippet: Analysis of Comet assay. Worms were treated with 5 μM CPT for 24 h and postincubated on new fresh NGM plates for various recovery times. Comet slides were prepared, lysed, treated with glyoxal, and electrophoresed in a neutral buffer. (A) Comet images were captured using an epifluorescence microscope (Carl Zeiss), Scale bar, 50 μm. (B) The tail moment of comets in (A) was analyzed using Comet Assay IV software. Comet slides were prepared, lysed, treated with RNase and proteinase K, and electrophoresed in a neutral buffer. (C) Comet images were captured using an epifluorescence microscope (Carl Zeiss), Scale bar, 50 μm. (D) The tail moment of comets in (C) was analyzed using Comet Assay IV software. no, no CPT-treated worms.

Techniques Used: Single Cell Gel Electrophoresis, Cycling Probe Technology, Microscopy, Software

50) Product Images from "Microglia Are Critical in Host Defense against Prion Disease"

Article Title: Microglia Are Critical in Host Defense against Prion Disease

Journal: Journal of Virology

doi: 10.1128/JVI.00549-18

Western blot and densitometry of PrPres in the brains of PLX5622-treated and untreated mice infected with scrapie. Mice infected with scrapie strain RML were treated with PLX5622 or untreated and euthanized at 80 dpi (A), at 100 dpi (B), or at clinical endpoint (terminal) (C). Brain homogenates were digested with proteinase K, and proteins were separated by SDS-PAGE and transferred to PVDF membranes. Blocked membranes were probed with anti-PrP (D13), and bands were visualized by chemiluminescence. (A and B) Approximately 5-s exposures to film using SuperSignal West Femto substrate; (C) 10-min exposure to film using ECL Western blotting substrate. The days postinfection of euthanasia of each mouse is given below the immunoblots in panel C. Densitometry on each lane was performed to assess differences between the groups, and plots of the adjusted volumes are shown to the right of the immunoblots. Statistical analysis was performed using a two-tailed  t  test comparing PLX5622-treated to untreated animals.  P  values are indicated.
Figure Legend Snippet: Western blot and densitometry of PrPres in the brains of PLX5622-treated and untreated mice infected with scrapie. Mice infected with scrapie strain RML were treated with PLX5622 or untreated and euthanized at 80 dpi (A), at 100 dpi (B), or at clinical endpoint (terminal) (C). Brain homogenates were digested with proteinase K, and proteins were separated by SDS-PAGE and transferred to PVDF membranes. Blocked membranes were probed with anti-PrP (D13), and bands were visualized by chemiluminescence. (A and B) Approximately 5-s exposures to film using SuperSignal West Femto substrate; (C) 10-min exposure to film using ECL Western blotting substrate. The days postinfection of euthanasia of each mouse is given below the immunoblots in panel C. Densitometry on each lane was performed to assess differences between the groups, and plots of the adjusted volumes are shown to the right of the immunoblots. Statistical analysis was performed using a two-tailed t test comparing PLX5622-treated to untreated animals. P values are indicated.

Techniques Used: Western Blot, Mouse Assay, Infection, SDS Page, Two Tailed Test

51) Product Images from "Topological and Mutational Analysis of Saccharomyces cerevisiae Ste14p, Founding Member of the Isoprenylcysteine Carboxyl Methyltransferase Family"

Article Title: Topological and Mutational Analysis of Saccharomyces cerevisiae Ste14p, Founding Member of the Isoprenylcysteine Carboxyl Methyltransferase Family

Journal: Molecular Biology of the Cell

doi:

Protease protection of Ste14p-myc Q3 and I239. Yeast membranes were treated with proteinase K in the presence and absence of 0.4% Triton X-100. After 5 min on ice, the reactions were terminated with 1 mM PMSF and 10% trichloroacetic acid. Samples were resolved by 12.5% SDS-PAGE and transferred to nitrocellulose. Ste14p-myc and Kar2p were detected with anti-myc and anti-Kar2p antisera, respectively. (A) SM3874 ( CEN STE14::myc Q3 ). (B) SM3876 ( CEN STE14::myc I239 ).
Figure Legend Snippet: Protease protection of Ste14p-myc Q3 and I239. Yeast membranes were treated with proteinase K in the presence and absence of 0.4% Triton X-100. After 5 min on ice, the reactions were terminated with 1 mM PMSF and 10% trichloroacetic acid. Samples were resolved by 12.5% SDS-PAGE and transferred to nitrocellulose. Ste14p-myc and Kar2p were detected with anti-myc and anti-Kar2p antisera, respectively. (A) SM3874 ( CEN STE14::myc Q3 ). (B) SM3876 ( CEN STE14::myc I239 ).

Techniques Used: SDS Page

Protease Protection of Ste14p-HA. (A) Topology model of Ste14p (6 span), with the site of the HA insertions denoted by a black circle. (B–E) Yeast membranes were treated with proteinase K in the presence and absence of 0.4% Triton X-100. After 5 min on ice, the reactions were terminated with 1 mM PMSF and 10% trichloroacetic acid. Samples were resolved by 12.5% SDS-PAGE and transferred to nitrocellulose. Ste14p-HA and Kar2p were detected with anti-HA and anti-Kar2p antisera, respectively. (B) SM3191 ( CEN STE14::HA V75 ) (C) SM3428 ( CEN STE14::HA K86 ) (D) SM4316 ( 2 μ STE14::HA K110 ) (E) SM3429 ( CEN STE14::HA G144 ).
Figure Legend Snippet: Protease Protection of Ste14p-HA. (A) Topology model of Ste14p (6 span), with the site of the HA insertions denoted by a black circle. (B–E) Yeast membranes were treated with proteinase K in the presence and absence of 0.4% Triton X-100. After 5 min on ice, the reactions were terminated with 1 mM PMSF and 10% trichloroacetic acid. Samples were resolved by 12.5% SDS-PAGE and transferred to nitrocellulose. Ste14p-HA and Kar2p were detected with anti-HA and anti-Kar2p antisera, respectively. (B) SM3191 ( CEN STE14::HA V75 ) (C) SM3428 ( CEN STE14::HA K86 ) (D) SM4316 ( 2 μ STE14::HA K110 ) (E) SM3429 ( CEN STE14::HA G144 ).

Techniques Used: SDS Page

52) Product Images from "Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) Isolates of Swine Origin Form Robust Biofilms"

Article Title: Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) Isolates of Swine Origin Form Robust Biofilms

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073376

Inhibition of biofilm formation by Proteinase K. Strains tested are shown along the x-axis and grouped based on methicillin-sensitivity and isolation source. The indicated strains were grown statically for 24 hours in media alone (- Prot. K) or in media supplemented with 100 µg/ml Proteinase K (+ Prot. K). Biofilm formation was quantified by standard microtiter assays and measuring the absorbance at 538 nm, plotted along the y-axis. Bars represent the average absorbance obtained from at least 3 independent plates representing biological replicates; error bars represent the SEM. Asterisks (*) denote a p -value less than 0.05 between the treated and untreated groups.
Figure Legend Snippet: Inhibition of biofilm formation by Proteinase K. Strains tested are shown along the x-axis and grouped based on methicillin-sensitivity and isolation source. The indicated strains were grown statically for 24 hours in media alone (- Prot. K) or in media supplemented with 100 µg/ml Proteinase K (+ Prot. K). Biofilm formation was quantified by standard microtiter assays and measuring the absorbance at 538 nm, plotted along the y-axis. Bars represent the average absorbance obtained from at least 3 independent plates representing biological replicates; error bars represent the SEM. Asterisks (*) denote a p -value less than 0.05 between the treated and untreated groups.

Techniques Used: Inhibition, Isolation

Dispersal of established biofilms by Proteinase K. Strains tested are shown along the x-axis and grouped based on methicillin-sensitivity and isolation source. The indicated strains were grown statically for 24 hours to allow biofilm formation. Wells were washed and treated with buffer alone (- Prot. K) or 100 µg/ml Proteinase K (+ Prot. K) for 2 hours. Biofilm formation was then quantified by standard microtiter assays and measuring the absorbance at 538 nm, plotted along the y-axis. Bars represent the average absorbance obtained from at least 3 independent plates representing biological replicates; error bars represent the SEM. Asterisks (*) denote a p -value less than 0.05 between the treated and untreated groups.
Figure Legend Snippet: Dispersal of established biofilms by Proteinase K. Strains tested are shown along the x-axis and grouped based on methicillin-sensitivity and isolation source. The indicated strains were grown statically for 24 hours to allow biofilm formation. Wells were washed and treated with buffer alone (- Prot. K) or 100 µg/ml Proteinase K (+ Prot. K) for 2 hours. Biofilm formation was then quantified by standard microtiter assays and measuring the absorbance at 538 nm, plotted along the y-axis. Bars represent the average absorbance obtained from at least 3 independent plates representing biological replicates; error bars represent the SEM. Asterisks (*) denote a p -value less than 0.05 between the treated and untreated groups.

Techniques Used: Isolation

53) Product Images from "ATM Functions at the Peroxisome to Induce Pexophagy in Response to ROS"

Article Title: ATM Functions at the Peroxisome to Induce Pexophagy in Response to ROS

Journal: Nature cell biology

doi: 10.1038/ncb3230

ATM kinase is localized at peroxisome and activated in response to ROS ( a ) Subcellular fractionation of HEK293 cells. Catalase and PMP70 were used as subcellular markers of the peroxisome (P). LDH, Lamin A/C and β-integrin were used as markers for cytosolic (C), nuclear (N) and membrane (M) fractions, respectively. WCE, whole cell extract. ( b ) Proteinase K assay in the presence or absence of Triton-X 100 performed with peroxisomal fractions obtained from HEK293 cells. Immunoblotting was performed with ATM, catalase and PMP70 antibodies. WCE, whole cell extract; P, peroxisome fraction. ( c ) HepG2 cells treated with H 2 O 2 (0.4 mM) at 1, 3, 6 h. Whole cell extracts (WCE) and peroxisomal fractions (P) were probed with the indicated antibodies. ( d ) Representative image of wild type (GM15871) and Zellweger (GM13267) fibroblasts treated with or without H 2 O 2 for 1 h and immunostained for active p-ATM (S1981) (green) and catalase (red). Scale bar, 15 μm. High-magnification images of boxed areas are indicated to the right (Scale bar, 5 μm). Uncropped images of western blots are shown in Supplementary Fig. S9 .
Figure Legend Snippet: ATM kinase is localized at peroxisome and activated in response to ROS ( a ) Subcellular fractionation of HEK293 cells. Catalase and PMP70 were used as subcellular markers of the peroxisome (P). LDH, Lamin A/C and β-integrin were used as markers for cytosolic (C), nuclear (N) and membrane (M) fractions, respectively. WCE, whole cell extract. ( b ) Proteinase K assay in the presence or absence of Triton-X 100 performed with peroxisomal fractions obtained from HEK293 cells. Immunoblotting was performed with ATM, catalase and PMP70 antibodies. WCE, whole cell extract; P, peroxisome fraction. ( c ) HepG2 cells treated with H 2 O 2 (0.4 mM) at 1, 3, 6 h. Whole cell extracts (WCE) and peroxisomal fractions (P) were probed with the indicated antibodies. ( d ) Representative image of wild type (GM15871) and Zellweger (GM13267) fibroblasts treated with or without H 2 O 2 for 1 h and immunostained for active p-ATM (S1981) (green) and catalase (red). Scale bar, 15 μm. High-magnification images of boxed areas are indicated to the right (Scale bar, 5 μm). Uncropped images of western blots are shown in Supplementary Fig. S9 .

Techniques Used: Fractionation, Western Blot

54) Product Images from "Mcl-1 and Bcl-xL sequestration of Bak confers differential resistance to BH3-only proteins"

Article Title: Mcl-1 and Bcl-xL sequestration of Bak confers differential resistance to BH3-only proteins

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-017-0010-6

MODE 2 complexes of Bak:Mcl-1 profoundly inhibit Bid signalling. a. Diagram showing interactions between the Bcl-2 family members in the tripartite mixtures tested. The BH3-only proteins Bid and Bid Bim induce similar activation (arrows) of the apoptotic effector Bak. The prosurvival protein Mcl-1 can sequester the BH3-only protein or activated Bak. Note that Bid binds poorly (dotted lines) to Mcl-1 (MODE 1 complex), allowing Mcl-1 to bind activated Bak (MODE 2 complex). b , c Mouse liver mitochondria (MLM) supplemented with c or without b Mcl-1 were treated with Bid or Bid Bim at the indicated concentrations. Mitochondrial cytochrome c release was assessed by western blot (WB) of supernatant (sup) and pellet (pel) fractions. Bak conformation change (activation) was assessed by incubation with proteinase K (PK) followed by separation to supernatant and pellet. Protein–protein interactions were determined by solubilising samples (pre-IP), immunoprecipitating (IP) for Mcl-1, and western blotting for Bak, HA (Bid or Bid Bim ) or Mcl-1. Samples in which Mcl-1 blocked the activation of Bak (MODE 1 inhibition, green) or blocked cytochrome c release by binding activated Bak (MODE 2 inhibition, blue) are highlighted. b and c ). # Long exposure of supernatant blots compared to pellets. *Bands on Mcl-1 blots are due to previous probing for HA.
Figure Legend Snippet: MODE 2 complexes of Bak:Mcl-1 profoundly inhibit Bid signalling. a. Diagram showing interactions between the Bcl-2 family members in the tripartite mixtures tested. The BH3-only proteins Bid and Bid Bim induce similar activation (arrows) of the apoptotic effector Bak. The prosurvival protein Mcl-1 can sequester the BH3-only protein or activated Bak. Note that Bid binds poorly (dotted lines) to Mcl-1 (MODE 1 complex), allowing Mcl-1 to bind activated Bak (MODE 2 complex). b , c Mouse liver mitochondria (MLM) supplemented with c or without b Mcl-1 were treated with Bid or Bid Bim at the indicated concentrations. Mitochondrial cytochrome c release was assessed by western blot (WB) of supernatant (sup) and pellet (pel) fractions. Bak conformation change (activation) was assessed by incubation with proteinase K (PK) followed by separation to supernatant and pellet. Protein–protein interactions were determined by solubilising samples (pre-IP), immunoprecipitating (IP) for Mcl-1, and western blotting for Bak, HA (Bid or Bid Bim ) or Mcl-1. Samples in which Mcl-1 blocked the activation of Bak (MODE 1 inhibition, green) or blocked cytochrome c release by binding activated Bak (MODE 2 inhibition, blue) are highlighted. b and c ). # Long exposure of supernatant blots compared to pellets. *Bands on Mcl-1 blots are due to previous probing for HA.

Techniques Used: Activation Assay, Western Blot, Incubation, Inhibition, Binding Assay

55) Product Images from "Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases"

Article Title: Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases

Journal: Parasites & Vectors

doi: 10.1186/s13071-018-2613-2

Promastigotes of L. donovani incubated with sand fly midguts dissected at different times post-blood meal and various controls. Leishmania donovani promastigotes (24 h after the releasing from macrophages) were incubated in a microtiter plate for 2 h with: a midguts of P. argentipes , P. orientalis , P. papatasi and S. schwetzi dissected at 6, 24, 32, 48 and 72 h post-blood meal; or b with saline, proteinase K (PK), rabbit blood, red cells of rabbit blood, human haemoglobin, blood + proteinase K, red cells + proteinase K and human haemoglobin + proteinase K. As a positive control, parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100 were used
Figure Legend Snippet: Promastigotes of L. donovani incubated with sand fly midguts dissected at different times post-blood meal and various controls. Leishmania donovani promastigotes (24 h after the releasing from macrophages) were incubated in a microtiter plate for 2 h with: a midguts of P. argentipes , P. orientalis , P. papatasi and S. schwetzi dissected at 6, 24, 32, 48 and 72 h post-blood meal; or b with saline, proteinase K (PK), rabbit blood, red cells of rabbit blood, human haemoglobin, blood + proteinase K, red cells + proteinase K and human haemoglobin + proteinase K. As a positive control, parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100 were used

Techniques Used: Incubation, Positive Control

56) Product Images from "Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases"

Article Title: Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases

Journal: Parasites & Vectors

doi: 10.1186/s13071-018-2613-2

Promastigotes of L. donovani incubated with sand fly midguts dissected at different times post-blood meal and various controls. Leishmania donovani promastigotes (24 h after the releasing from macrophages) were incubated in a microtiter plate for 2 h with: a midguts of P. argentipes , P. orientalis , P. papatasi and S. schwetzi dissected at 6, 24, 32, 48 and 72 h post-blood meal; or b with saline, proteinase K (PK), rabbit blood, red cells of rabbit blood, human haemoglobin, blood + proteinase K, red cells + proteinase K and human haemoglobin + proteinase K. As a positive control, parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100 were used
Figure Legend Snippet: Promastigotes of L. donovani incubated with sand fly midguts dissected at different times post-blood meal and various controls. Leishmania donovani promastigotes (24 h after the releasing from macrophages) were incubated in a microtiter plate for 2 h with: a midguts of P. argentipes , P. orientalis , P. papatasi and S. schwetzi dissected at 6, 24, 32, 48 and 72 h post-blood meal; or b with saline, proteinase K (PK), rabbit blood, red cells of rabbit blood, human haemoglobin, blood + proteinase K, red cells + proteinase K and human haemoglobin + proteinase K. As a positive control, parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100 were used

Techniques Used: Incubation, Positive Control

57) Product Images from "BRCA2 regulates DMC1-mediated recombination through the BRC repeats"

Article Title: BRCA2 regulates DMC1-mediated recombination through the BRC repeats

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1601691113

Purified GFP-MBP-BRCA2 complements  Brca2 -deficient cells and stimulates RAD51-mediated joint molecule formation. ( A ) BRCA2 tagged with GFP-MBP at the N terminus was purified from human HEK293 cells and analyzed by SDS/PAGE. Lane 1: BRCA2 (0.9 μg) was loaded on a precast 7.5% SDS/PAGE gel and stained with SYPRO Ruby. Lane 2: Western blot of purified BRCA2 protein (0.5 μg) using an antibody specific for the carboxy-terminus of BRCA2 (CA1033, EMD).  M r , size markers. ( B ) Mitomycin C survival of stably transfected clones of  Brca2 -deficient hamster cells complemented with human GFP-MBP-tagged BRCA2 (green and blue), the vector containing the GFP-MBP tag (violet and pink), V79 parental cells ( Brca2 +/+ ) (orange) and VC8 ( Brca2 −/− ) (gold). ( C ) RAD51 (75 nM) and the indicated concentrations of BRCA2 were preincubated with a 5′-end  32 P-labeled 90-mer ssDNA [2.4 nM (molecule)] for 10 min at 37 °C and scDNA [0.8 nM (molecule)] was added last to start the reaction. The mix was incubated at 37 °C for 30 min, terminated by incubation with Proteinase K, and resolved on a 1% agarose gel. ( D ) Quantification of  C . Error bars in  D  represent the SD for three independent experiments.
Figure Legend Snippet: Purified GFP-MBP-BRCA2 complements Brca2 -deficient cells and stimulates RAD51-mediated joint molecule formation. ( A ) BRCA2 tagged with GFP-MBP at the N terminus was purified from human HEK293 cells and analyzed by SDS/PAGE. Lane 1: BRCA2 (0.9 μg) was loaded on a precast 7.5% SDS/PAGE gel and stained with SYPRO Ruby. Lane 2: Western blot of purified BRCA2 protein (0.5 μg) using an antibody specific for the carboxy-terminus of BRCA2 (CA1033, EMD). M r , size markers. ( B ) Mitomycin C survival of stably transfected clones of Brca2 -deficient hamster cells complemented with human GFP-MBP-tagged BRCA2 (green and blue), the vector containing the GFP-MBP tag (violet and pink), V79 parental cells ( Brca2 +/+ ) (orange) and VC8 ( Brca2 −/− ) (gold). ( C ) RAD51 (75 nM) and the indicated concentrations of BRCA2 were preincubated with a 5′-end 32 P-labeled 90-mer ssDNA [2.4 nM (molecule)] for 10 min at 37 °C and scDNA [0.8 nM (molecule)] was added last to start the reaction. The mix was incubated at 37 °C for 30 min, terminated by incubation with Proteinase K, and resolved on a 1% agarose gel. ( D ) Quantification of C . Error bars in D represent the SD for three independent experiments.

Techniques Used: Purification, SDS Page, Staining, Western Blot, Stable Transfection, Transfection, Clone Assay, Plasmid Preparation, Labeling, Incubation, Agarose Gel Electrophoresis

58) Product Images from "Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie"

Article Title: Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200421083

Time course of PrP Sc  deposition in muscle tissue. Western blot detection of PrP27-30 extracted from different muscles and sciatic nerve of hamsters orally challenged with 263K scrapie and sacrificed at the following time points after infection. ( A ) At 100 days after infection, ( B – D ) 130 days after infection, ( E ) onset of clinical signs for scrapie (139–149 days after infection), and ( F ) at the terminal stage of disease (161–173 days after infection). Lanes with test samples: M1, M. biceps femoris (hindlimb); M2, M. tibialis cranialis (hindlimb); M3, M. triceps brachii (forelimb); M4, M. extensor carpi radialis (forelimb); M5, M. trapezius (shoulder); M6, M. masseter (head); M7, M. psoas major (back); T, tongue; H, heart; SN, sciatic nerve. Lanes with control samples: 1, proteinase K–digested brain homogenate from terminally ill scrapie hamsters containing 1 ∞ 10 –7  g ( B  and  F ) or 5 ∞ 10 –7  g ( A ,  C – E ) brain tissue; 2, skeletal muscle from an uninfected hamster spiked before extraction with 5 ∞ 10 –6  g ( A ,  B , and  F ), 1 ∞ 10 –5  g ( C  and  D ), or 2 ∞ 10 –5  g ( E ) brain homogenate from terminally ill scrapie hamsters; 3, skeletal muscle from a mock-challenged hamster sacrificed at 173 days after infection ( F ). For each stage of incubation representative results are shown. ( G ) Time scale displaying the mean incubation period and the preclinical and clinical phases of incubation of hamsters orally infected with 263K scrapie. Small vertical arrows indicate time points at which animals were screened for PrP Sc  in muscles. dai, days after infection.
Figure Legend Snippet: Time course of PrP Sc deposition in muscle tissue. Western blot detection of PrP27-30 extracted from different muscles and sciatic nerve of hamsters orally challenged with 263K scrapie and sacrificed at the following time points after infection. ( A ) At 100 days after infection, ( B – D ) 130 days after infection, ( E ) onset of clinical signs for scrapie (139–149 days after infection), and ( F ) at the terminal stage of disease (161–173 days after infection). Lanes with test samples: M1, M. biceps femoris (hindlimb); M2, M. tibialis cranialis (hindlimb); M3, M. triceps brachii (forelimb); M4, M. extensor carpi radialis (forelimb); M5, M. trapezius (shoulder); M6, M. masseter (head); M7, M. psoas major (back); T, tongue; H, heart; SN, sciatic nerve. Lanes with control samples: 1, proteinase K–digested brain homogenate from terminally ill scrapie hamsters containing 1 ∞ 10 –7 g ( B and F ) or 5 ∞ 10 –7 g ( A , C – E ) brain tissue; 2, skeletal muscle from an uninfected hamster spiked before extraction with 5 ∞ 10 –6 g ( A , B , and F ), 1 ∞ 10 –5 g ( C and D ), or 2 ∞ 10 –5 g ( E ) brain homogenate from terminally ill scrapie hamsters; 3, skeletal muscle from a mock-challenged hamster sacrificed at 173 days after infection ( F ). For each stage of incubation representative results are shown. ( G ) Time scale displaying the mean incubation period and the preclinical and clinical phases of incubation of hamsters orally infected with 263K scrapie. Small vertical arrows indicate time points at which animals were screened for PrP Sc in muscles. dai, days after infection.

Techniques Used: Western Blot, Infection, Incubation

59) Product Images from "Transgenic Rabbits Expressing Ovine PrP Are Susceptible to ScrapieTransgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates"

Article Title: Transgenic Rabbits Expressing Ovine PrP Are Susceptible to ScrapieTransgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005077

Brain PrP Sc in ovine PrP transgenic rabbit infected with LA21K fast scrapie prions. (a) Western blot analyses of the brain from wild-type and tgOv rabbits mock-infected or inoculated with LA21K fast prions for the presence of proteinase K (PK)-resistant PrP Sc . The equivalent of 2 mg brain tissue were loaded in lane 1–6, 3 mg in lanes 7 and 8. (b) Electrophoretic pattern and (c) glycoform ratios (plotted as means ± SEM(c)) of PrP res in the brains and spleens of tgOv rabbit and tg338 mice infected with LA21K fast prions. The same amount of brain (0.5 mg) and spleen (3 mg) tissue equivalent was loaded on the gel.
Figure Legend Snippet: Brain PrP Sc in ovine PrP transgenic rabbit infected with LA21K fast scrapie prions. (a) Western blot analyses of the brain from wild-type and tgOv rabbits mock-infected or inoculated with LA21K fast prions for the presence of proteinase K (PK)-resistant PrP Sc . The equivalent of 2 mg brain tissue were loaded in lane 1–6, 3 mg in lanes 7 and 8. (b) Electrophoretic pattern and (c) glycoform ratios (plotted as means ± SEM(c)) of PrP res in the brains and spleens of tgOv rabbit and tg338 mice infected with LA21K fast prions. The same amount of brain (0.5 mg) and spleen (3 mg) tissue equivalent was loaded on the gel.

Techniques Used: Transgenic Assay, Infection, Western Blot, Mouse Assay

60) Product Images from "RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome"

Article Title: RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

Journal: Genome Biology

doi: 10.1186/gb-2014-15-1-r3

Overview of the PIP-seq method. (A) In the PIP-seq method, cells are cross-linked with formaldehyde or 254-nm UV light, or not cross-linked. They are lysed and divided into footprint and RNase digestion control samples. The footprint sample is treated with an RNase (ss- or dsRNase), which results in a population of RNase-protected RNA–RBP complexes. The protein cross-links are then reversed (by heating for formaldehyde cross-links or by proteinase K treatment for UV cross-links), leaving only the footprints where the RNA was protein-bound. For the RNase digestion control sample, which is designed to control for RNase insensitive regions, the order of operations is reversed; bound proteins are first removed by treatment with SDS and proteinase K, and then the unprotected RNA sample is subjected to RNase treatment. Strand-specific high-throughput sequencing libraries are prepared from both footprint and RNase digestion control samples and normalized using rehybridization and duplex-specific nuclease (DSN) treatment. PPSs are identified from the sequencing data using a Poisson model. Screenshots show UCSC browser views of sequencing reads from the footprint and RNase digestion control sample (same scale) and PPSs identified from the regions of the genes listed. (B,C) Absolute distribution of PPSs throughout RNA species for formaldehyde (B) and UV (C) cross-linked PIP-seq experiments. (D,E) Average PPS count per RNA molecule (classified by RNA type (mRNA and lncRNA) and transcript region (for example, 5′ UTR)) for formaldehyde (D) and UV (E) cross-linked PIP-seq experiments. Percentages indicate the fraction of each RNA type or region that contains PPS information. (F) Average expression ( y -axis) of human mRNAs separated by total number of PPSs identified in their sequence ( x -axis) for PPSs identified using formaldehyde cross-linking. CDS, coding sequence; DSN, duplex-specific nuclease; dsRNase, double-stranded RNase; lncRNA, long non-coding RNA; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase; UTR, untranslated region.
Figure Legend Snippet: Overview of the PIP-seq method. (A) In the PIP-seq method, cells are cross-linked with formaldehyde or 254-nm UV light, or not cross-linked. They are lysed and divided into footprint and RNase digestion control samples. The footprint sample is treated with an RNase (ss- or dsRNase), which results in a population of RNase-protected RNA–RBP complexes. The protein cross-links are then reversed (by heating for formaldehyde cross-links or by proteinase K treatment for UV cross-links), leaving only the footprints where the RNA was protein-bound. For the RNase digestion control sample, which is designed to control for RNase insensitive regions, the order of operations is reversed; bound proteins are first removed by treatment with SDS and proteinase K, and then the unprotected RNA sample is subjected to RNase treatment. Strand-specific high-throughput sequencing libraries are prepared from both footprint and RNase digestion control samples and normalized using rehybridization and duplex-specific nuclease (DSN) treatment. PPSs are identified from the sequencing data using a Poisson model. Screenshots show UCSC browser views of sequencing reads from the footprint and RNase digestion control sample (same scale) and PPSs identified from the regions of the genes listed. (B,C) Absolute distribution of PPSs throughout RNA species for formaldehyde (B) and UV (C) cross-linked PIP-seq experiments. (D,E) Average PPS count per RNA molecule (classified by RNA type (mRNA and lncRNA) and transcript region (for example, 5′ UTR)) for formaldehyde (D) and UV (E) cross-linked PIP-seq experiments. Percentages indicate the fraction of each RNA type or region that contains PPS information. (F) Average expression ( y -axis) of human mRNAs separated by total number of PPSs identified in their sequence ( x -axis) for PPSs identified using formaldehyde cross-linking. CDS, coding sequence; DSN, duplex-specific nuclease; dsRNase, double-stranded RNase; lncRNA, long non-coding RNA; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase; UTR, untranslated region.

Techniques Used: Next-Generation Sequencing, Sequencing, Expressing

61) Product Images from "RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome"

Article Title: RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

Journal: Genome Biology

doi: 10.1186/gb-2014-15-1-r3

Overview of the PIP-seq method. (A) In the PIP-seq method, cells are cross-linked with formaldehyde or 254-nm UV light, or not cross-linked. They are lysed and divided into footprint and RNase digestion control samples. The footprint sample is treated with an RNase (ss- or dsRNase), which results in a population of RNase-protected RNA–RBP complexes. The protein cross-links are then reversed (by heating for formaldehyde cross-links or by proteinase K treatment for UV cross-links), leaving only the footprints where the RNA was protein-bound. For the RNase digestion control sample, which is designed to control for RNase insensitive regions, the order of operations is reversed; bound proteins are first removed by treatment with SDS and proteinase K, and then the unprotected RNA sample is subjected to RNase treatment. Strand-specific high-throughput sequencing libraries are prepared from both footprint and RNase digestion control samples and normalized using rehybridization and duplex-specific nuclease (DSN) treatment. PPSs are identified from the sequencing data using a Poisson model. Screenshots show UCSC browser views of sequencing reads from the footprint and RNase digestion control sample (same scale) and PPSs identified from the regions of the genes listed. (B,C) Absolute distribution of PPSs throughout RNA species for formaldehyde (B) and UV (C) cross-linked PIP-seq experiments. (D,E) Average PPS count per RNA molecule (classified by RNA type (mRNA and lncRNA) and transcript region (for example, 5′ UTR)) for formaldehyde (D) and UV (E) cross-linked PIP-seq experiments. Percentages indicate the fraction of each RNA type or region that contains PPS information. (F) Average expression ( y -axis) of human mRNAs separated by total number of PPSs identified in their sequence ( x -axis) for PPSs identified using formaldehyde cross-linking. CDS, coding sequence; DSN, duplex-specific nuclease; dsRNase, double-stranded RNase; lncRNA, long non-coding RNA; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase; UTR, untranslated region.
Figure Legend Snippet: Overview of the PIP-seq method. (A) In the PIP-seq method, cells are cross-linked with formaldehyde or 254-nm UV light, or not cross-linked. They are lysed and divided into footprint and RNase digestion control samples. The footprint sample is treated with an RNase (ss- or dsRNase), which results in a population of RNase-protected RNA–RBP complexes. The protein cross-links are then reversed (by heating for formaldehyde cross-links or by proteinase K treatment for UV cross-links), leaving only the footprints where the RNA was protein-bound. For the RNase digestion control sample, which is designed to control for RNase insensitive regions, the order of operations is reversed; bound proteins are first removed by treatment with SDS and proteinase K, and then the unprotected RNA sample is subjected to RNase treatment. Strand-specific high-throughput sequencing libraries are prepared from both footprint and RNase digestion control samples and normalized using rehybridization and duplex-specific nuclease (DSN) treatment. PPSs are identified from the sequencing data using a Poisson model. Screenshots show UCSC browser views of sequencing reads from the footprint and RNase digestion control sample (same scale) and PPSs identified from the regions of the genes listed. (B,C) Absolute distribution of PPSs throughout RNA species for formaldehyde (B) and UV (C) cross-linked PIP-seq experiments. (D,E) Average PPS count per RNA molecule (classified by RNA type (mRNA and lncRNA) and transcript region (for example, 5′ UTR)) for formaldehyde (D) and UV (E) cross-linked PIP-seq experiments. Percentages indicate the fraction of each RNA type or region that contains PPS information. (F) Average expression ( y -axis) of human mRNAs separated by total number of PPSs identified in their sequence ( x -axis) for PPSs identified using formaldehyde cross-linking. CDS, coding sequence; DSN, duplex-specific nuclease; dsRNase, double-stranded RNase; lncRNA, long non-coding RNA; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase; UTR, untranslated region.

Techniques Used: Next-Generation Sequencing, Sequencing, Expressing

62) Product Images from "Transglutaminase-mediated Intramolecular Cross-linking of Membrane-bound ?-Synuclein Promotes Amyloid Formation in Lewy Bodies *"

Article Title: Transglutaminase-mediated Intramolecular Cross-linking of Membrane-bound ?-Synuclein Promotes Amyloid Formation in Lewy Bodies *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.033969

γ-Glutamyl-ϵ-lysine cross-links in brain fractions accumulate in α-synuclein immunopositive chaotrope-insoluble inclusions/Lewy bodies.  The relative abundance of GGEL was determined by enzymatic hydrolysis of sample proteins in  18 O-water and comparison with light internal GGEL standard by mass spectrometry. GGEL was related to total amino acid content.  A , GGEL accumulation associates with regions affected by degenerative changes in human brain specimen affected by Lewy body dementia ( LBD ), Alzheimer disease, or in age-matched controls ( CTRL ). Over 50% of total tissue GGEL content was recovered by either anti-ubiquitin or anti-α-synuclein affinity isolation from chaotrope-, detergent-, and thiol-insoluble brain proteins ( B ), indicating that most of the cross-linked inclusions expose both antigens in AD and LBD. Lewy bodies were exhaustively digested with proteinase K. Residual proteins remaining after digestions were dissolved in guanidine HCl and co-precipitated with a different protein (egg albumin) to disrupt β-sheet amyloid arrays between proteins. The GGEL density in proteinase K-resistant fractions is by an order of magnitude lower than in the proteinase-digestible part ( C ).  Bars  represent the means of duplicate determinations from three different donors.  Asterisks  and  hatch marks  indicate significant ( p
Figure Legend Snippet: γ-Glutamyl-ϵ-lysine cross-links in brain fractions accumulate in α-synuclein immunopositive chaotrope-insoluble inclusions/Lewy bodies. The relative abundance of GGEL was determined by enzymatic hydrolysis of sample proteins in 18 O-water and comparison with light internal GGEL standard by mass spectrometry. GGEL was related to total amino acid content. A , GGEL accumulation associates with regions affected by degenerative changes in human brain specimen affected by Lewy body dementia ( LBD ), Alzheimer disease, or in age-matched controls ( CTRL ). Over 50% of total tissue GGEL content was recovered by either anti-ubiquitin or anti-α-synuclein affinity isolation from chaotrope-, detergent-, and thiol-insoluble brain proteins ( B ), indicating that most of the cross-linked inclusions expose both antigens in AD and LBD. Lewy bodies were exhaustively digested with proteinase K. Residual proteins remaining after digestions were dissolved in guanidine HCl and co-precipitated with a different protein (egg albumin) to disrupt β-sheet amyloid arrays between proteins. The GGEL density in proteinase K-resistant fractions is by an order of magnitude lower than in the proteinase-digestible part ( C ). Bars represent the means of duplicate determinations from three different donors. Asterisks and hatch marks indicate significant ( p

Techniques Used: Mass Spectrometry, Isolation

63) Product Images from "Superparamagnetic Nanoparticle Capture of Prions for Amplification ▿"

Article Title: Superparamagnetic Nanoparticle Capture of Prions for Amplification ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02451-10

Binding of PrP Sc to MagnaBind superparamagnetic beads. Bound PrP Sc was detected by proteinase K digestion and anti-PrP (6D11) immunoblot. (A) RML scrapie-infected mouse brain homogenate was incubated with MagnaBind (Mag) or Dynal (Dyn) magnetic beads
Figure Legend Snippet: Binding of PrP Sc to MagnaBind superparamagnetic beads. Bound PrP Sc was detected by proteinase K digestion and anti-PrP (6D11) immunoblot. (A) RML scrapie-infected mouse brain homogenate was incubated with MagnaBind (Mag) or Dynal (Dyn) magnetic beads

Techniques Used: Binding Assay, Infection, Incubation, Magnetic Beads

64) Product Images from "A30P ?-Synuclein interferes with the stable integration of adult-born neurons into the olfactory network"

Article Title: A30P ?-Synuclein interferes with the stable integration of adult-born neurons into the olfactory network

Journal: Scientific Reports

doi: 10.1038/srep03931

Olfactory bulb pathology in A30P α-SYN mice. (a) Immunofluorescent micrographs showing the overexpression pattern of human A30P α-SYN (15G7, red) under control of the Thy1-promoter in the OB of 6-month-old transgenic mice: z-stack projections of confocal series. Scale bar - 50 μm. (b–e) Representative OB sections immunostained for phosphorylated α-SYN (pSer129, DAB), a marker for aberrant protein modification. Scale bars - 50 μm. (b) Distribution of phosphorylated α-SYN in (c) PGNs, (d) MCs and (e) GCs. Note that only MCs are positive for pSer129. (f–j) Representative OB sections with immunostaining for fibrillar protein, detected by Proteinase K digestion and human α-SYN specific antibody (15G7, DAB). Scale bars - 50 μm. Images with higher magnification of the (g) glomerular, (h) MC and (i) GC layer. Some α-SYN aggregates were detected in MCs of transgenic, but not wild-type, mice. (j) Olfactory discrimination task. Six-month-old Control (wild-type) ( n = 8) and A30P α-SYN mice ( n = 7) were exposed to different binary odour mixtures (e.g. 55% S + and 45% S − vs . 45% S + and 55% S − ). Mice were trained to dig in a bowl containing more of S + odour. Correct choices for S + were plotted against the different mixture pairs. Note that with increasing similarity of the mixtures, the performance of A30P α-SYN mice dropped compared to Control. (k) Olfactory memory task. ** p
Figure Legend Snippet: Olfactory bulb pathology in A30P α-SYN mice. (a) Immunofluorescent micrographs showing the overexpression pattern of human A30P α-SYN (15G7, red) under control of the Thy1-promoter in the OB of 6-month-old transgenic mice: z-stack projections of confocal series. Scale bar - 50 μm. (b–e) Representative OB sections immunostained for phosphorylated α-SYN (pSer129, DAB), a marker for aberrant protein modification. Scale bars - 50 μm. (b) Distribution of phosphorylated α-SYN in (c) PGNs, (d) MCs and (e) GCs. Note that only MCs are positive for pSer129. (f–j) Representative OB sections with immunostaining for fibrillar protein, detected by Proteinase K digestion and human α-SYN specific antibody (15G7, DAB). Scale bars - 50 μm. Images with higher magnification of the (g) glomerular, (h) MC and (i) GC layer. Some α-SYN aggregates were detected in MCs of transgenic, but not wild-type, mice. (j) Olfactory discrimination task. Six-month-old Control (wild-type) ( n = 8) and A30P α-SYN mice ( n = 7) were exposed to different binary odour mixtures (e.g. 55% S + and 45% S − vs . 45% S + and 55% S − ). Mice were trained to dig in a bowl containing more of S + odour. Correct choices for S + were plotted against the different mixture pairs. Note that with increasing similarity of the mixtures, the performance of A30P α-SYN mice dropped compared to Control. (k) Olfactory memory task. ** p

Techniques Used: Mouse Assay, Over Expression, Transgenic Assay, Marker, Modification, Immunostaining

65) Product Images from "Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface"

Article Title: Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191622

Expression and surface-accessibility of Hbp-β domain fusions. (A) Coomassie-stained SDS-PAGE of whole cell lysates (c) and culture supernatants (m) of MC1061 cells expressing wild-type Hbp and Hbp( Spe I). (B) Coomassie-stained gel of whole cell lysates (c) and culture supernatants (m) of MC1061 (left panel) or MC1061 degP ::S210A (right panel) expressing Hbp-β-domain fusions. (C) Coomassie-stained gel of whole cell lysates of cells of MC1061 expressing the Hbp-β-domain fusions incubated with proteinase K, either for 60 min on ice (0°) or 30 min at 37 °C (37°). Included are also untreated controls (-).The positions of bands representing unprocessed Hbp passenger-β-domain fusions (*), and processed Hbp passenger (●) and Hbpβ (▲) are indicated. The latter are only detected for Hbp( Spe I). In panel C, the prominent ~79-kDa of Hbp-Δβcleavage degradation product is indicated by (#), the position of the proteinase K bands is indicated by the closed arrowhead, whereas the open arrowheads indicate the control bands used for densitometric analysis.
Figure Legend Snippet: Expression and surface-accessibility of Hbp-β domain fusions. (A) Coomassie-stained SDS-PAGE of whole cell lysates (c) and culture supernatants (m) of MC1061 cells expressing wild-type Hbp and Hbp( Spe I). (B) Coomassie-stained gel of whole cell lysates (c) and culture supernatants (m) of MC1061 (left panel) or MC1061 degP ::S210A (right panel) expressing Hbp-β-domain fusions. (C) Coomassie-stained gel of whole cell lysates of cells of MC1061 expressing the Hbp-β-domain fusions incubated with proteinase K, either for 60 min on ice (0°) or 30 min at 37 °C (37°). Included are also untreated controls (-).The positions of bands representing unprocessed Hbp passenger-β-domain fusions (*), and processed Hbp passenger (●) and Hbpβ (▲) are indicated. The latter are only detected for Hbp( Spe I). In panel C, the prominent ~79-kDa of Hbp-Δβcleavage degradation product is indicated by (#), the position of the proteinase K bands is indicated by the closed arrowhead, whereas the open arrowheads indicate the control bands used for densitometric analysis.

Techniques Used: Expressing, Staining, SDS Page, Incubation

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Centrifugation:

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Amplification:

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Filtration:

Article Title: Comparative Proteome Analysis of Multi-Layer Cocoon of the Silkworm, Bombyx mori
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Real-time Polymerase Chain Reaction:

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Incubation:

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Article Title: Calorie restriction increases telomerase activity, enhances autophagy, and improves diastolic dysfunction in diabetic rat hearts
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Article Title: Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescence
Article Snippet: De-crosslinking was performed by the addition of 30 μL of protease K (Roche) at 65 °C overnight. .. To remove residual RNA, 15 μL of RNaseA cocktail (Ambion) was added to the samples and incubated at 37 °C for 45 min. DNA was recovered by adding 7 mL of isopropanol and 70 μL of NucleoMag 96 PCR beads (Bioke) and incubated for 30 min at room temperature.

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Article Snippet: Antibody incubation time was 32 min. For EGFR, uPAR and TF and 24 min. For TF. .. Antigen retrieval for uPAR was done with protease K (Ventana-Roche, CA, USA) for 8 min followed by heating at 100 °C with cell conditioning 1 (CC1, Ventana-Roche, CA, USA) buffer for 16 min. For CK, TF and EGFR standard heat induced epitope retrieval (32 min, 100 °C) in CC1 buffer was used.

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Formalin-fixed Paraffin-Embedded:

Article Title: Tissue-Specific Effects of Reduced β-catenin Expression on Adenomatous Polyposis Coli Mutation-Instigated Tumorigenesis in Mouse Colon and Ovarian Epithelium
Article Snippet: .. Terminal deoxynucleotidyl transferase ( T dT)-mediated d U TP n ick e nd-labeling (TUNEL) assay To assess apoptosis, TUNEL assays were undertaken using 4-μm sections of formalin-fixed, paraffin-embedded mouse colon tissues, after the tissue sections were deparaffinized, rehydrated and treated with 20 μg/ml protease K (Roche Applied Sciences, Indianapolis, IN) at 37°C for 15 min. .. The nicked DNA was labeled by using terminal transferase (TdT) (New England Biolabs, Ipswich, MA) and Biotin-16-UTP (Roche Applied Sciences) according to the manufacturer’s recommendation.

Activity Assay:

Article Title: Two Nested Developmental Waves Demarcate a Compartment Boundary in the Mouse Lung
Article Snippet: The following day, the sections were washed with TBST for 2 hours at room temperature and the alkaline phosphatase activity was detected with nitro-blue tetrazolium (N6639, Sigma) and 5-bromo-4-chloro-3′-indolyphosphate (B6149, Sigma). .. At room temperature, the lungs were rehydrated through a methanol/DEPC-PBS (diethylpyrocarbonate treated PBS) gradient, permeabilized with 20 ug mL−1 protease K (03115887001, Roche) in DEPC-PBS for 10 minutes and fixed with PBS with 0.25% glutaraldehyde and 4% PFA for 20 minutes.

Article Title: Comparative Proteome Analysis of Multi-Layer Cocoon of the Silkworm, Bombyx mori
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Protein Misfolding Cyclic Amplification:

Article Title: Relative Impact of Complement Receptors CD21/35 (Cr2/1) on Scrapie Pathogenesis in Mice
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Western Blot:

Article Title: A Novel PGC-1? Isoform in Brain Localizes to Mitochondria and Associates with PINK1 and VDAC
Article Snippet: Purified mitochondria were incubated with protease K and mitochondrial pellet was lysed in a buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 1% NP-40, and a mixture of protease inhibitors (Roche Molecular Biochemicals) and phosphatase inhibitors (Sigma). .. Co-immunoprecipitates were collected and used for Western blot analysis.

Article Title: Relative Impact of Complement Receptors CD21/35 (Cr2/1) on Scrapie Pathogenesis in Mice
Article Snippet: .. We assessed the presence of PrPSc in 10% (wt/vol) homogenate after protease K (Roche) digestion (10 µg/ml for spleen and 50 µg/ml for brain) and Western blotting using anti-PrP monoclonal antibody (Ab) BAR 224 (Cayman Chemical) conjugated to horseradish peroxidase (HRP). .. Blots were developed using chemiluminescent substrates hydrogen peroxide and luminol for 5 min at room temperature and visualized using a GE digital imager and ImageQuant software.

Hybridization:

Article Title: Two Nested Developmental Waves Demarcate a Compartment Boundary in the Mouse Lung
Article Snippet: After incubation with hybridization solution (50% formamide, 5x SSC (sodium chloride and sodium citrate), 5X Denhardt’s solution, 500 ug uL−1 salmon sperm DNA, 250 ug uL−1 yeast tRNA) for 1 hour at room temperature, the sections were incubated with 1 ug mL−1 riboprobes diluted in hybridization solution in a slide oven (24100, Boekel) at 62°C overnight. .. At room temperature, the lungs were rehydrated through a methanol/DEPC-PBS (diethylpyrocarbonate treated PBS) gradient, permeabilized with 20 ug mL−1 protease K (03115887001, Roche) in DEPC-PBS for 10 minutes and fixed with PBS with 0.25% glutaraldehyde and 4% PFA for 20 minutes.

TUNEL Assay:

Article Title: Tissue-Specific Effects of Reduced β-catenin Expression on Adenomatous Polyposis Coli Mutation-Instigated Tumorigenesis in Mouse Colon and Ovarian Epithelium
Article Snippet: .. Terminal deoxynucleotidyl transferase ( T dT)-mediated d U TP n ick e nd-labeling (TUNEL) assay To assess apoptosis, TUNEL assays were undertaken using 4-μm sections of formalin-fixed, paraffin-embedded mouse colon tissues, after the tissue sections were deparaffinized, rehydrated and treated with 20 μg/ml protease K (Roche Applied Sciences, Indianapolis, IN) at 37°C for 15 min. .. The nicked DNA was labeled by using terminal transferase (TdT) (New England Biolabs, Ipswich, MA) and Biotin-16-UTP (Roche Applied Sciences) according to the manufacturer’s recommendation.

Immunohistochemistry:

Article Title: Urokinase-type plasminogen activator receptor (uPAR), tissue factor (TF) and epidermal growth factor receptor (EGFR): tumor expression patterns and prognostic value in oral cancer
Article Snippet: Paragraph title: IHC staining ... Antigen retrieval for uPAR was done with protease K (Ventana-Roche, CA, USA) for 8 min followed by heating at 100 °C with cell conditioning 1 (CC1, Ventana-Roche, CA, USA) buffer for 16 min. For CK, TF and EGFR standard heat induced epitope retrieval (32 min, 100 °C) in CC1 buffer was used.

Ligation:

Article Title: Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescence
Article Snippet: Again, the ligation efficiency was examined on agarose gel. .. De-crosslinking was performed by the addition of 30 μL of protease K (Roche) at 65 °C overnight.

Protease Inhibitor:

Article Title: Comparative Proteome Analysis of Multi-Layer Cocoon of the Silkworm, Bombyx mori
Article Snippet: .. For protease inhibitor activity assays, four proteases were used as target proteases: trypsin (Sigma, T1426), chymotrypsin (Sigma, C4129), elastase (Sigma, 45124), and protease K (Roche, 11060325). ..

Polymerase Chain Reaction:

Article Title: The antidepressant action of 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid is mediated by phosphorylation of histone deacetylase 5
Article Snippet: Finally, the protein-DNA cross-links were incubated with protease K (Roche Applied Sciences) for 2 h at 42℃ and immunoprecipitated DNA was purified. .. Purified DNA samples were normalized and subjected to PCR analysis.

Article Title: Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescence
Article Snippet: De-crosslinking was performed by the addition of 30 μL of protease K (Roche) at 65 °C overnight. .. To remove residual RNA, 15 μL of RNaseA cocktail (Ambion) was added to the samples and incubated at 37 °C for 45 min. DNA was recovered by adding 7 mL of isopropanol and 70 μL of NucleoMag 96 PCR beads (Bioke) and incubated for 30 min at room temperature.

Article Title: Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
Article Snippet: Then, we added 1 μl protease K 50 ng μl−1 (Roche), and incubated the mixture at 37 °C for 1 h, followed by 95 °C for 5 min, to inactivate the protease. .. An extra DNAse treatment (Turbo DNA-free kit, Ambion, Life Technologies) was performed to reduce gDNA contamination. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for RT–quantitative PCR (Thermo Scientific), following manufacturer's instructions.

Sonication:

Article Title: The antidepressant action of 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid is mediated by phosphorylation of histone deacetylase 5
Article Snippet: The sonicated samples were rotated to preclear with protein A agarose (Roche Applied Sciences) for 1 h at 4℃ and anti-HDAC5 antibody (Abcam) was added and incubated overnight at 4℃ followed by incubation with fresh Protein A agarose for 2 h at 4℃. .. Finally, the protein-DNA cross-links were incubated with protease K (Roche Applied Sciences) for 2 h at 42℃ and immunoprecipitated DNA was purified.

Binding Assay:

Article Title: Prophenoloxidase-Mediated Ex Vivo Immunity to Delay Fungal Infection after Insect Ecdysis
Article Snippet: .. To remove spore surface proteins, spores were suspended and incubated in the binding buffer containing 0.5 µg/µL Protease K (Roche) at room temperature for 12 h. High concentration of HCl can hydrolyzate proteins ( ). .. In order to remove all proteins, spores were also incubated in 37% HCl at room temperature for 12 h. To remove chitin component, spores were suspended and incubated in the binding buffer (200 µL) containing 1 µg/µL chitinase (Roche) and 1-mM phenylmethylsulfonyl fluoride (PMSF) (to inhibit protease contamination in this product according to our assay).

DNA Extraction:

Article Title: Diagnosis for choroideremia in a large Chinese pedigree by next-generation sequencing (NGS) and non-invasive prenatal testing (NIPT)
Article Snippet: Methods for DNA extraction from the peripheral blood of associated family members and the amniotic fluid from the fetus were described previously ( , , ). .. The filtrate was discarded, 1 ml Nucleic Lysis Buffer, 100 µl 20% SDS and 10 µl Protease K (20 mg/ml; Roche Diagnostics, Indianapolis, IN, USA) were added, and the mixture was incubated at 56°C for 4 h. An equivalent volume of phenol was added, mixed, and centrifuged for 10 min at 1,600 × g .

Article Title: FoxO1 signaling plays a pivotal role in the cardiac telomere biology responses to calorie restriction
Article Snippet: Extraction of genomic DNA from mouse tissues Mouse tissue samples were lysed by incubation at 55 °C for 48 h in 200 μL lysis buffer containing 10 mM Tris/HCl (pH 8.0), 0.1 mM EDTA (pH 8.0), 2 % sodium dodecyl sulfate (SDS), and 500 μg/mL protease K (Roche Diagnostic, Tokyo, Japan). .. Genomic DNA extraction was performed using a DNeasy Tissue Kit (Qiagen K.K.,Tokyo, Japan) according to the manufacturer’s recommendations, as described previously [ ].

Article Title: Calorie restriction increases telomerase activity, enhances autophagy, and improves diastolic dysfunction in diabetic rat hearts
Article Snippet: .. Genomic DNA extraction Rat tissue samples were lysed by incubation at 55 °C for 48 h in 200 ul lysis buffer containing 10 mmol/l Tris HCl (pH 8.0), 0.1 mmol/l EDTA (pH 8.0), 2 % SDS, and 500 g/ml protease K (Roche Diagnostic, Tokyo, Japan). .. Genomic DNA extraction was performed using a DNeasy tissue kit (Qiagen, Tokyo, Japan), according to a previous publication [ ].

Article Title: Detection of Coxiella burnetii in Ambient Air after a Large Q Fever Outbreak
Article Snippet: Next, 400 μl protease K (20 mg/ml; Roche Diagnostics Nederland B.V, Almere, the Netherlands) was added and incubated for 10 minutes at 55°C. .. To each tube, 50 μl Bacillus thuringiensis spore suspension (dilution 1:10; Raven Labs, Omaha, USA) was added to serve as an internal control for both DNA extraction and qPCR amplification.

Article Title: Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
Article Snippet: Briefly, the flies were macerated using pellet pestles and homogenized in 100 μl DNA extraction buffer (1 M Tris-HCl at pH 8.2, 0.5 M EDTA, 5 M NaCl). .. Then, we added 1 μl protease K 50 ng μl−1 (Roche), and incubated the mixture at 37 °C for 1 h, followed by 95 °C for 5 min, to inactivate the protease.

Labeling:

Article Title: Tissue-Specific Effects of Reduced β-catenin Expression on Adenomatous Polyposis Coli Mutation-Instigated Tumorigenesis in Mouse Colon and Ovarian Epithelium
Article Snippet: Terminal deoxynucleotidyl transferase ( T dT)-mediated d U TP n ick e nd-labeling (TUNEL) assay To assess apoptosis, TUNEL assays were undertaken using 4-μm sections of formalin-fixed, paraffin-embedded mouse colon tissues, after the tissue sections were deparaffinized, rehydrated and treated with 20 μg/ml protease K (Roche Applied Sciences, Indianapolis, IN) at 37°C for 15 min. .. The nicked DNA was labeled by using terminal transferase (TdT) (New England Biolabs, Ipswich, MA) and Biotin-16-UTP (Roche Applied Sciences) according to the manufacturer’s recommendation.

Purification:

Article Title: A Novel PGC-1? Isoform in Brain Localizes to Mitochondria and Associates with PINK1 and VDAC
Article Snippet: .. Purified mitochondria were incubated with protease K and mitochondrial pellet was lysed in a buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 1% NP-40, and a mixture of protease inhibitors (Roche Molecular Biochemicals) and phosphatase inhibitors (Sigma). .. After homogenizing with 20 strokes by using a Dounce homogenizer (Bellco Glass), mitochondrial lysates were centrifuged at 20,000 × g, and the supernatants were used for co-immunoprecipitation assay using the Pierce co-immunoprecipitation kit.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
Article Snippet: Then, we added 1 μl protease K 50 ng μl−1 (Roche), and incubated the mixture at 37 °C for 1 h, followed by 95 °C for 5 min, to inactivate the protease. .. The material used for the RT–PCR experiments described in was obtained from 15 virgin males aged between 3–7 days, and was macerated using pellet pestles and homogenized in 500 μl TRI Reagent and centrifuged at 12,000g for 1 min, to lower tissue debris.

Lysis:

Article Title: Diagnosis for choroideremia in a large Chinese pedigree by next-generation sequencing (NGS) and non-invasive prenatal testing (NIPT)
Article Snippet: .. The filtrate was discarded, 1 ml Nucleic Lysis Buffer, 100 µl 20% SDS and 10 µl Protease K (20 mg/ml; Roche Diagnostics, Indianapolis, IN, USA) were added, and the mixture was incubated at 56°C for 4 h. An equivalent volume of phenol was added, mixed, and centrifuged for 10 min at 1,600 × g . ..

Article Title: FoxO1 signaling plays a pivotal role in the cardiac telomere biology responses to calorie restriction
Article Snippet: .. Extraction of genomic DNA from mouse tissues Mouse tissue samples were lysed by incubation at 55 °C for 48 h in 200 μL lysis buffer containing 10 mM Tris/HCl (pH 8.0), 0.1 mM EDTA (pH 8.0), 2 % sodium dodecyl sulfate (SDS), and 500 μg/mL protease K (Roche Diagnostic, Tokyo, Japan). .. Genomic DNA extraction was performed using a DNeasy Tissue Kit (Qiagen K.K.,Tokyo, Japan) according to the manufacturer’s recommendations, as described previously [ ].

Article Title: Calorie restriction increases telomerase activity, enhances autophagy, and improves diastolic dysfunction in diabetic rat hearts
Article Snippet: .. Genomic DNA extraction Rat tissue samples were lysed by incubation at 55 °C for 48 h in 200 ul lysis buffer containing 10 mmol/l Tris HCl (pH 8.0), 0.1 mmol/l EDTA (pH 8.0), 2 % SDS, and 500 g/ml protease K (Roche Diagnostic, Tokyo, Japan). .. Genomic DNA extraction was performed using a DNeasy tissue kit (Qiagen, Tokyo, Japan), according to a previous publication [ ].

Concentration Assay:

Article Title: Prophenoloxidase-Mediated Ex Vivo Immunity to Delay Fungal Infection after Insect Ecdysis
Article Snippet: .. To remove spore surface proteins, spores were suspended and incubated in the binding buffer containing 0.5 µg/µL Protease K (Roche) at room temperature for 12 h. High concentration of HCl can hydrolyzate proteins ( ). .. In order to remove all proteins, spores were also incubated in 37% HCl at room temperature for 12 h. To remove chitin component, spores were suspended and incubated in the binding buffer (200 µL) containing 1 µg/µL chitinase (Roche) and 1-mM phenylmethylsulfonyl fluoride (PMSF) (to inhibit protease contamination in this product according to our assay).

Mouse Assay:

Article Title: Relative Impact of Complement Receptors CD21/35 (Cr2/1) on Scrapie Pathogenesis in Mice
Article Snippet: We assessed the presence of PrPSc in 10% (wt/vol) homogenate after protease K (Roche) digestion (10 µg/ml for spleen and 50 µg/ml for brain) and Western blotting using anti-PrP monoclonal antibody (Ab) BAR 224 (Cayman Chemical) conjugated to horseradish peroxidase (HRP). .. Briefly, we used 10% normal brain homogenate (NBH) in PMCA buffer (PBS, 1% Triton X-100, 4 mM EDTA, 150 mM NaCl) from PrPC -overexpressing transgenic mice of strain Tga20 ( ) as the substrate for amplification of previously undetectable PrPSc .

Chromatin Immunoprecipitation:

Article Title: The antidepressant action of 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid is mediated by phosphorylation of histone deacetylase 5
Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) assay ... Finally, the protein-DNA cross-links were incubated with protease K (Roche Applied Sciences) for 2 h at 42℃ and immunoprecipitated DNA was purified.

In Situ Hybridization:

Article Title: Two Nested Developmental Waves Demarcate a Compartment Boundary in the Mouse Lung
Article Snippet: Paragraph title: Section and whole mount in situ hybridization ... At room temperature, the lungs were rehydrated through a methanol/DEPC-PBS (diethylpyrocarbonate treated PBS) gradient, permeabilized with 20 ug mL−1 protease K (03115887001, Roche) in DEPC-PBS for 10 minutes and fixed with PBS with 0.25% glutaraldehyde and 4% PFA for 20 minutes.

Plasmid Preparation:

Article Title: Tissue-Specific Effects of Reduced β-catenin Expression on Adenomatous Polyposis Coli Mutation-Instigated Tumorigenesis in Mouse Colon and Ovarian Epithelium
Article Snippet: Terminal deoxynucleotidyl transferase ( T dT)-mediated d U TP n ick e nd-labeling (TUNEL) assay To assess apoptosis, TUNEL assays were undertaken using 4-μm sections of formalin-fixed, paraffin-embedded mouse colon tissues, after the tissue sections were deparaffinized, rehydrated and treated with 20 μg/ml protease K (Roche Applied Sciences, Indianapolis, IN) at 37°C for 15 min. .. Terminal deoxynucleotidyl transferase ( T dT)-mediated d U TP n ick e nd-labeling (TUNEL) assay To assess apoptosis, TUNEL assays were undertaken using 4-μm sections of formalin-fixed, paraffin-embedded mouse colon tissues, after the tissue sections were deparaffinized, rehydrated and treated with 20 μg/ml protease K (Roche Applied Sciences, Indianapolis, IN) at 37°C for 15 min.

Software:

Article Title: Relative Impact of Complement Receptors CD21/35 (Cr2/1) on Scrapie Pathogenesis in Mice
Article Snippet: We assessed the presence of PrPSc in 10% (wt/vol) homogenate after protease K (Roche) digestion (10 µg/ml for spleen and 50 µg/ml for brain) and Western blotting using anti-PrP monoclonal antibody (Ab) BAR 224 (Cayman Chemical) conjugated to horseradish peroxidase (HRP). .. Blots were developed using chemiluminescent substrates hydrogen peroxide and luminol for 5 min at room temperature and visualized using a GE digital imager and ImageQuant software.

Co-Immunoprecipitation Assay:

Article Title: A Novel PGC-1? Isoform in Brain Localizes to Mitochondria and Associates with PINK1 and VDAC
Article Snippet: Paragraph title: 2.7. Co-immunoprecipitation assay ... Purified mitochondria were incubated with protease K and mitochondrial pellet was lysed in a buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 1% NP-40, and a mixture of protease inhibitors (Roche Molecular Biochemicals) and phosphatase inhibitors (Sigma).

Agarose Gel Electrophoresis:

Article Title: Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescence
Article Snippet: Again, the ligation efficiency was examined on agarose gel. .. De-crosslinking was performed by the addition of 30 μL of protease K (Roche) at 65 °C overnight.

Transgenic Assay:

Article Title: Relative Impact of Complement Receptors CD21/35 (Cr2/1) on Scrapie Pathogenesis in Mice
Article Snippet: We assessed the presence of PrPSc in 10% (wt/vol) homogenate after protease K (Roche) digestion (10 µg/ml for spleen and 50 µg/ml for brain) and Western blotting using anti-PrP monoclonal antibody (Ab) BAR 224 (Cayman Chemical) conjugated to horseradish peroxidase (HRP). .. Briefly, we used 10% normal brain homogenate (NBH) in PMCA buffer (PBS, 1% Triton X-100, 4 mM EDTA, 150 mM NaCl) from PrPC -overexpressing transgenic mice of strain Tga20 ( ) as the substrate for amplification of previously undetectable PrPSc .

Immunoprecipitation:

Article Title: The antidepressant action of 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid is mediated by phosphorylation of histone deacetylase 5
Article Snippet: .. Finally, the protein-DNA cross-links were incubated with protease K (Roche Applied Sciences) for 2 h at 42℃ and immunoprecipitated DNA was purified. .. Purified DNA samples were normalized and subjected to PCR analysis.

Staining:

Article Title: Urokinase-type plasminogen activator receptor (uPAR), tissue factor (TF) and epidermal growth factor receptor (EGFR): tumor expression patterns and prognostic value in oral cancer
Article Snippet: Paragraph title: IHC staining ... Antigen retrieval for uPAR was done with protease K (Ventana-Roche, CA, USA) for 8 min followed by heating at 100 °C with cell conditioning 1 (CC1, Ventana-Roche, CA, USA) buffer for 16 min. For CK, TF and EGFR standard heat induced epitope retrieval (32 min, 100 °C) in CC1 buffer was used.

Permeability:

Article Title: Two Nested Developmental Waves Demarcate a Compartment Boundary in the Mouse Lung
Article Snippet: For some riboprobes, E17 lungs were sliced at 200~300 um thickness orthogonal to the main bronchi of the left and right caudal lobes with a micro-scissor to increase permeability. .. At room temperature, the lungs were rehydrated through a methanol/DEPC-PBS (diethylpyrocarbonate treated PBS) gradient, permeabilized with 20 ug mL−1 protease K (03115887001, Roche) in DEPC-PBS for 10 minutes and fixed with PBS with 0.25% glutaraldehyde and 4% PFA for 20 minutes.

Cross-linking Immunoprecipitation:

Article Title: Relative Impact of Complement Receptors CD21/35 (Cr2/1) on Scrapie Pathogenesis in Mice
Article Snippet: After euthanasia, the following samples were collected and frozen or fixed in 4% formaldehyde in PBS: serum, spleen (half fixed, half frozen), kidneys (one fixed, one frozen), tail clip, and brain (half fixed, half frozen). .. We assessed the presence of PrPSc in 10% (wt/vol) homogenate after protease K (Roche) digestion (10 µg/ml for spleen and 50 µg/ml for brain) and Western blotting using anti-PrP monoclonal antibody (Ab) BAR 224 (Cayman Chemical) conjugated to horseradish peroxidase (HRP).

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  • 80
    Roche desaturase disruption buffer
    Substrate utilization of a Thraustrochytrium delta-4 <t>desaturase.</t> A membrane preparation from yeast cells expressing Thraustrochytrium delta-4 desaturase was tested for CoA linked acyl-desaturation (panels a , b ) and phosphatidylcholine linked acyl-desaturation (panels c , d ). In panels a showing substrate depletion, and b showing product accumulation, the membrane preparations were preincubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]22:5n-3-CoA substrate and NADH (time point zero) and further incubation for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]22:5n-3-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for times as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )
    Desaturase Disruption Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Roche hela k tmem170a gfp
    Downregulation of <t>TMEM170A</t> alters ER structure and induces tubular ER proliferation. (A–E) TMEM170A siRNA alters ER structure in <t>HeLa</t> K cells. Immunofluorescence analysis using antibodies to calnexin (A; green), LEM4 (B; green) or RTN4/NogoA+B (C,D; green) in combination with other nuclear envelope markers (as indicated; red) reveals that, in silenced cells, the ER is concentrated in a restricted area close to one side of the nucleus or is atypically widely spread and, additionally, ER aggregation is observed (white arrowheads; A,D) compared with controls. TMEM170A-silenced cells stained with anti-CLIMP-63 antibody (E; red) showed a reduced ER sheet signal, relative to control cells. Nuclei in blue. Scale bars: 10 μm. (F) TEM of the ER and nuclear envelope of control (Fa) and TMEM170A-silenced cells (Fb–e). TMEM170A silencing induces nuclear envelope invagination (white arrowheads, b) and increased tubular ER (red arrowheads, Fc; another example at higher magnification in Fd). Connections between nuclear envelope and the membranes were visible (Fc, light blue arrowhead). Occasionally, ER tubules were seen connected with part of semi-organized smooth ER (cisternae, purple arrowheads) and rarely whorls (white stars) (Fe). Scale bars: 1 μm (black) or 2 μm (white). Section thickness: 60 nm.
    Hela K Tmem170a Gfp, supplied by Roche, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche proteinase k
    RCA can detect rSDS-PrP Sc oligomers at early stages of the disease. a Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [ 16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels. b Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above. c To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of <t>proteinase</t> K and processed as described in the legend to Fig. 4a . The asterisk in b and c indicates the position of oligomer traces
    Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche ypd k media
    Glucose starvation promotes efficient polarized cell growth and induces the expression of mating-related genes in MTL a/a cells of C . albicans . (A) Morphologies of the laboratory strain GH1013 ( MTL a / a ) grown on <t>YPD-K</t> and YP-K media. 1 × 10 5 cells were spotted on YPD-K and YP-K media and cultured at 25°C for five days. Scale bar for colonies (left panels), 2 mm; scale bar for cells (right panels), 10 μm. (B) Morphologies of three clinical C . albicans strains ( MTL a / a ) grown on YP-K medium at 30°C for four days. Scale bar for colonies (inset), 2 mm; scale bar for cells, 10 μm. (C) Relative expression levels of mating-related genes normalized to ACT1 . Cells of GH1013 were used, and culture conditions were same as described in panel (A). Error bars represent standard errors of technical duplicates. * p
    Ypd K Media, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Substrate utilization of a Thraustrochytrium delta-4 desaturase. A membrane preparation from yeast cells expressing Thraustrochytrium delta-4 desaturase was tested for CoA linked acyl-desaturation (panels a , b ) and phosphatidylcholine linked acyl-desaturation (panels c , d ). In panels a showing substrate depletion, and b showing product accumulation, the membrane preparations were preincubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]22:5n-3-CoA substrate and NADH (time point zero) and further incubation for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]22:5n-3-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for times as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )

    Journal: Lipids

    Article Title: Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

    doi: 10.1007/s11745-017-4235-4

    Figure Lengend Snippet: Substrate utilization of a Thraustrochytrium delta-4 desaturase. A membrane preparation from yeast cells expressing Thraustrochytrium delta-4 desaturase was tested for CoA linked acyl-desaturation (panels a , b ) and phosphatidylcholine linked acyl-desaturation (panels c , d ). In panels a showing substrate depletion, and b showing product accumulation, the membrane preparations were preincubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]22:5n-3-CoA substrate and NADH (time point zero) and further incubation for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]22:5n-3-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for times as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )

    Article Snippet: After 24 h, yeast cells were harvested, washed with 25 mM Tris–HCl, pH 7.6, and resuspended in Desaturase Disruption Buffer (0.1 M potassium phosphate pH 7.2, 0.33 M sucrose, 1 mg/ml BSA, 4 mM NADH, 4000 U/ml catalase) or Elongase Disruption Buffer (20 mM Tris–HCl, pH 7.9, 10 mM MgCl2 , 1 mM EDTA, 5% (v/v) glycerol, 0.3 M ammonium sulfate) containing protease inhibitor (Complete, Roche Applied Science).

    Techniques: Expressing, Incubation, Standard Deviation

    Demonstration of in vitro desaturase activities of delta-6 desaturase from Ostreococcus tauri (d6Des( Ot )), lane 1 panel a , delta-5 desaturase from Thraustochytrium sp. (d5Des( Tc )), lane 1 panel b , and delta-4 desaturase from Pavlova lutheri (d4Des( Pl )), lane 1 panel c , in membrane preparations of yeast cells heterologously expressing these enzymes. Duplicate reactions are presented in each lane . Control assays were performed with membrane fractions isolated from yeast strains harboring an empty vector (Control), lane 2 in panels a – c . The figure depicts autoradiographic images of TLC plates of separated [ 14 C]methyl esters (ME) prepared from the total lipids extracted from the assays containing an [ 14 C]acyl-CoA (d6Des( Ot ), 18:2n-6; d5Des( Tc ), 20:3n-6; and d4Des( Pl ), 22:5n-3) and NADH. The conversion of [ 14 C]substrate-fatty acid to [ 14 C]product-fatty acid is shown for the desaturases as an average of the conversion in duplicate assays (% conversion). All enzyme assays shown in this figure were performed without the addition of lysoPtdCho. Corresponding incubations when lysoPtdCho was added to the assay resulted in the following substrate to product conversions: d6Des( Ot ), 3%; d5Des( Tc ), 3%; d4Des( Pl ), 2%

    Journal: Lipids

    Article Title: Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

    doi: 10.1007/s11745-017-4235-4

    Figure Lengend Snippet: Demonstration of in vitro desaturase activities of delta-6 desaturase from Ostreococcus tauri (d6Des( Ot )), lane 1 panel a , delta-5 desaturase from Thraustochytrium sp. (d5Des( Tc )), lane 1 panel b , and delta-4 desaturase from Pavlova lutheri (d4Des( Pl )), lane 1 panel c , in membrane preparations of yeast cells heterologously expressing these enzymes. Duplicate reactions are presented in each lane . Control assays were performed with membrane fractions isolated from yeast strains harboring an empty vector (Control), lane 2 in panels a – c . The figure depicts autoradiographic images of TLC plates of separated [ 14 C]methyl esters (ME) prepared from the total lipids extracted from the assays containing an [ 14 C]acyl-CoA (d6Des( Ot ), 18:2n-6; d5Des( Tc ), 20:3n-6; and d4Des( Pl ), 22:5n-3) and NADH. The conversion of [ 14 C]substrate-fatty acid to [ 14 C]product-fatty acid is shown for the desaturases as an average of the conversion in duplicate assays (% conversion). All enzyme assays shown in this figure were performed without the addition of lysoPtdCho. Corresponding incubations when lysoPtdCho was added to the assay resulted in the following substrate to product conversions: d6Des( Ot ), 3%; d5Des( Tc ), 3%; d4Des( Pl ), 2%

    Article Snippet: After 24 h, yeast cells were harvested, washed with 25 mM Tris–HCl, pH 7.6, and resuspended in Desaturase Disruption Buffer (0.1 M potassium phosphate pH 7.2, 0.33 M sucrose, 1 mg/ml BSA, 4 mM NADH, 4000 U/ml catalase) or Elongase Disruption Buffer (20 mM Tris–HCl, pH 7.9, 10 mM MgCl2 , 1 mM EDTA, 5% (v/v) glycerol, 0.3 M ammonium sulfate) containing protease inhibitor (Complete, Roche Applied Science).

    Techniques: In Vitro, Expressing, Isolation, Plasmid Preparation, Thin Layer Chromatography

    Substrate utilization of a Phytophthora infestans omega-3 desaturase. A membrane preparation from yeast cells expressing Phytophthora infestans omega-3 desaturase was tested for CoA linked acyl-desaturation (panels a , b ) and phosphatidylcholine linked acyl-desaturation (panels c , d ). In panels a , showing substrate depletion, and b showing product accumulation, the membrane preparations were pre-incubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]20:4n-6-CoA substrate and NADH (time point zero in the graph) and further incubated for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]20:4n-6-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for times as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )

    Journal: Lipids

    Article Title: Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

    doi: 10.1007/s11745-017-4235-4

    Figure Lengend Snippet: Substrate utilization of a Phytophthora infestans omega-3 desaturase. A membrane preparation from yeast cells expressing Phytophthora infestans omega-3 desaturase was tested for CoA linked acyl-desaturation (panels a , b ) and phosphatidylcholine linked acyl-desaturation (panels c , d ). In panels a , showing substrate depletion, and b showing product accumulation, the membrane preparations were pre-incubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]20:4n-6-CoA substrate and NADH (time point zero in the graph) and further incubated for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]20:4n-6-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for times as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )

    Article Snippet: After 24 h, yeast cells were harvested, washed with 25 mM Tris–HCl, pH 7.6, and resuspended in Desaturase Disruption Buffer (0.1 M potassium phosphate pH 7.2, 0.33 M sucrose, 1 mg/ml BSA, 4 mM NADH, 4000 U/ml catalase) or Elongase Disruption Buffer (20 mM Tris–HCl, pH 7.9, 10 mM MgCl2 , 1 mM EDTA, 5% (v/v) glycerol, 0.3 M ammonium sulfate) containing protease inhibitor (Complete, Roche Applied Science).

    Techniques: Expressing, Incubation, Standard Deviation

    Substrate utilization of the S. cerevisiae delta-9 desaturase (d9Des( Sc )). A membrane preparation from yeast cells overexpressing the endogenous delta-9 desaturase was tested for its ability to use acyl-CoA (panels a , b ) and phosphatidylcholine-linked (panels c , d ) fatty acid substrates. In panels a showing substrate depletion, and b showing product accumulation, the membrane preparations were pre-incubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]16:0-CoA substrate and NADH (time point zero in the graph) and further incubated for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]16:0-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for time periods as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )

    Journal: Lipids

    Article Title: Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

    doi: 10.1007/s11745-017-4235-4

    Figure Lengend Snippet: Substrate utilization of the S. cerevisiae delta-9 desaturase (d9Des( Sc )). A membrane preparation from yeast cells overexpressing the endogenous delta-9 desaturase was tested for its ability to use acyl-CoA (panels a , b ) and phosphatidylcholine-linked (panels c , d ) fatty acid substrates. In panels a showing substrate depletion, and b showing product accumulation, the membrane preparations were pre-incubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]16:0-CoA substrate and NADH (time point zero in the graph) and further incubated for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]16:0-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for time periods as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )

    Article Snippet: After 24 h, yeast cells were harvested, washed with 25 mM Tris–HCl, pH 7.6, and resuspended in Desaturase Disruption Buffer (0.1 M potassium phosphate pH 7.2, 0.33 M sucrose, 1 mg/ml BSA, 4 mM NADH, 4000 U/ml catalase) or Elongase Disruption Buffer (20 mM Tris–HCl, pH 7.9, 10 mM MgCl2 , 1 mM EDTA, 5% (v/v) glycerol, 0.3 M ammonium sulfate) containing protease inhibitor (Complete, Roche Applied Science).

    Techniques: Incubation, Standard Deviation

    Substrate utilization of a Phytophthora sojae delta-12 desaturase. A membrane preparation from yeast cells expressing Phytophthora sojae delta-12 desaturase was tested for CoA linked acyl-desaturation (panels a , b ) and phosphatidylcholine linked acyl-desaturation (panels c , d ). In panels a , showing substrate depletion, and b showing product accumulation, the membrane preparations were pre-incubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]18:1n-9-CoA substrate and NADH (time point zero in the graph) and further incubated for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]18:1n-9-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for times as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )

    Journal: Lipids

    Article Title: Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

    doi: 10.1007/s11745-017-4235-4

    Figure Lengend Snippet: Substrate utilization of a Phytophthora sojae delta-12 desaturase. A membrane preparation from yeast cells expressing Phytophthora sojae delta-12 desaturase was tested for CoA linked acyl-desaturation (panels a , b ) and phosphatidylcholine linked acyl-desaturation (panels c , d ). In panels a , showing substrate depletion, and b showing product accumulation, the membrane preparations were pre-incubated with DTNB and 20:1n-9-CoA for 10 min and the desaturase reaction was initiated by adding [ 14 C]18:1n-9-CoA substrate and NADH (time point zero in the graph) and further incubated for times as shown in the figure. In panels c showing substrate depletion, and d showing product accumulation, the membrane preparations were pre-incubated with [ 14 C]18:1n-9-CoA and lysophosphatidylcholine for 15 min and the desaturase reaction was initiated by addition of NADH and further incubated for times as shown in the figure. The results presented are from triplicate assays with the standard deviation as error bars . In each panel [ 14 C]substrates and [ 14 C]products were followed as: free fatty acids ( unfilled squares , FFA); phosphatidylcholine ( unfilled diamonds , PtdCho); and acyl-CoA ( unfilled triangles )

    Article Snippet: After 24 h, yeast cells were harvested, washed with 25 mM Tris–HCl, pH 7.6, and resuspended in Desaturase Disruption Buffer (0.1 M potassium phosphate pH 7.2, 0.33 M sucrose, 1 mg/ml BSA, 4 mM NADH, 4000 U/ml catalase) or Elongase Disruption Buffer (20 mM Tris–HCl, pH 7.9, 10 mM MgCl2 , 1 mM EDTA, 5% (v/v) glycerol, 0.3 M ammonium sulfate) containing protease inhibitor (Complete, Roche Applied Science).

    Techniques: Expressing, Incubation, Standard Deviation

    A pathway involving two elongation steps and five desaturation steps allows production of EPA (20:5n-3) and DHA (22:6n-3) from oleic acid (OLA, 18:1n-9). In this pathway seven desaturase enzymes (Des) and three elongase enzymes (Elo), further described in Table 1 , modify fatty acid substrates that are covalently bound to a carrier (e.g. coenzyme A or phosphatidylcholine). Shown is a simplified route for synthesis of DHA that only describes a fatty acid moiety, but not the acyl-carrier, that is recognized by each enzyme

    Journal: Lipids

    Article Title: Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

    doi: 10.1007/s11745-017-4235-4

    Figure Lengend Snippet: A pathway involving two elongation steps and five desaturation steps allows production of EPA (20:5n-3) and DHA (22:6n-3) from oleic acid (OLA, 18:1n-9). In this pathway seven desaturase enzymes (Des) and three elongase enzymes (Elo), further described in Table 1 , modify fatty acid substrates that are covalently bound to a carrier (e.g. coenzyme A or phosphatidylcholine). Shown is a simplified route for synthesis of DHA that only describes a fatty acid moiety, but not the acyl-carrier, that is recognized by each enzyme

    Article Snippet: After 24 h, yeast cells were harvested, washed with 25 mM Tris–HCl, pH 7.6, and resuspended in Desaturase Disruption Buffer (0.1 M potassium phosphate pH 7.2, 0.33 M sucrose, 1 mg/ml BSA, 4 mM NADH, 4000 U/ml catalase) or Elongase Disruption Buffer (20 mM Tris–HCl, pH 7.9, 10 mM MgCl2 , 1 mM EDTA, 5% (v/v) glycerol, 0.3 M ammonium sulfate) containing protease inhibitor (Complete, Roche Applied Science).

    Techniques:

    Demonstration of in vitro desaturase activities of delta-12 desaturase from Phytophthora sojae (d12Des( Ps )), lane 1 in panel a, omega-3 desaturases from Pythium irregulare (o3Des( Pir )), lane 1 in panel b , and Phytophthora infestans (o3Des( Pi )), lane 2 in panel b , and delta-4 desaturase from Thraustochytrium sp. (d4Des( Tc )), lane 2 in panel c . Duplicate reactions are presented in each lane . The activities were measured in membrane preparations of yeast cells heterologously expressing these enzymes. Control assays were performed with membrane fractions isolated from yeast strains harboring an empty vector (Control), lane 2 in panel a , lane 3 in panel b and lane 1 in panel c . The figure depicts autoradiographic images of TLC plates of separated [ 14 C]methyl esters (ME) prepared from the total lipids extracted from assays containing an [ 14 C]acyl-CoA(d12Des( Ps ), 18:1n-9; o3Des’s, 20:4n-6; and d4Des( Tc ), 22:5n-3) and NADH. The conversion of [ 14 C]substrate-fatty acid to [ 14 C]product-fatty acid is shown for the desaturases as an average of the conversion in duplicate assays (% conversion). Lysophosphatidylcholine (lysoPtdCho) was added to each of the assays shown in this figure. Corresponding incubations without the addition of lysoPtdCho gave the following substrate conversions to product: d12Des( Ps ), 6%; o3Des( Pir ), 6%; o3Des( Pi ), 8%; and d4Des( Tc ), 20%

    Journal: Lipids

    Article Title: Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

    doi: 10.1007/s11745-017-4235-4

    Figure Lengend Snippet: Demonstration of in vitro desaturase activities of delta-12 desaturase from Phytophthora sojae (d12Des( Ps )), lane 1 in panel a, omega-3 desaturases from Pythium irregulare (o3Des( Pir )), lane 1 in panel b , and Phytophthora infestans (o3Des( Pi )), lane 2 in panel b , and delta-4 desaturase from Thraustochytrium sp. (d4Des( Tc )), lane 2 in panel c . Duplicate reactions are presented in each lane . The activities were measured in membrane preparations of yeast cells heterologously expressing these enzymes. Control assays were performed with membrane fractions isolated from yeast strains harboring an empty vector (Control), lane 2 in panel a , lane 3 in panel b and lane 1 in panel c . The figure depicts autoradiographic images of TLC plates of separated [ 14 C]methyl esters (ME) prepared from the total lipids extracted from assays containing an [ 14 C]acyl-CoA(d12Des( Ps ), 18:1n-9; o3Des’s, 20:4n-6; and d4Des( Tc ), 22:5n-3) and NADH. The conversion of [ 14 C]substrate-fatty acid to [ 14 C]product-fatty acid is shown for the desaturases as an average of the conversion in duplicate assays (% conversion). Lysophosphatidylcholine (lysoPtdCho) was added to each of the assays shown in this figure. Corresponding incubations without the addition of lysoPtdCho gave the following substrate conversions to product: d12Des( Ps ), 6%; o3Des( Pir ), 6%; o3Des( Pi ), 8%; and d4Des( Tc ), 20%

    Article Snippet: After 24 h, yeast cells were harvested, washed with 25 mM Tris–HCl, pH 7.6, and resuspended in Desaturase Disruption Buffer (0.1 M potassium phosphate pH 7.2, 0.33 M sucrose, 1 mg/ml BSA, 4 mM NADH, 4000 U/ml catalase) or Elongase Disruption Buffer (20 mM Tris–HCl, pH 7.9, 10 mM MgCl2 , 1 mM EDTA, 5% (v/v) glycerol, 0.3 M ammonium sulfate) containing protease inhibitor (Complete, Roche Applied Science).

    Techniques: In Vitro, Expressing, Isolation, Plasmid Preparation, Thin Layer Chromatography

    Downregulation of TMEM170A alters ER structure and induces tubular ER proliferation. (A–E) TMEM170A siRNA alters ER structure in HeLa K cells. Immunofluorescence analysis using antibodies to calnexin (A; green), LEM4 (B; green) or RTN4/NogoA+B (C,D; green) in combination with other nuclear envelope markers (as indicated; red) reveals that, in silenced cells, the ER is concentrated in a restricted area close to one side of the nucleus or is atypically widely spread and, additionally, ER aggregation is observed (white arrowheads; A,D) compared with controls. TMEM170A-silenced cells stained with anti-CLIMP-63 antibody (E; red) showed a reduced ER sheet signal, relative to control cells. Nuclei in blue. Scale bars: 10 μm. (F) TEM of the ER and nuclear envelope of control (Fa) and TMEM170A-silenced cells (Fb–e). TMEM170A silencing induces nuclear envelope invagination (white arrowheads, b) and increased tubular ER (red arrowheads, Fc; another example at higher magnification in Fd). Connections between nuclear envelope and the membranes were visible (Fc, light blue arrowhead). Occasionally, ER tubules were seen connected with part of semi-organized smooth ER (cisternae, purple arrowheads) and rarely whorls (white stars) (Fe). Scale bars: 1 μm (black) or 2 μm (white). Section thickness: 60 nm.

    Journal: Journal of Cell Science

    Article Title: Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    doi: 10.1242/jcs.175273

    Figure Lengend Snippet: Downregulation of TMEM170A alters ER structure and induces tubular ER proliferation. (A–E) TMEM170A siRNA alters ER structure in HeLa K cells. Immunofluorescence analysis using antibodies to calnexin (A; green), LEM4 (B; green) or RTN4/NogoA+B (C,D; green) in combination with other nuclear envelope markers (as indicated; red) reveals that, in silenced cells, the ER is concentrated in a restricted area close to one side of the nucleus or is atypically widely spread and, additionally, ER aggregation is observed (white arrowheads; A,D) compared with controls. TMEM170A-silenced cells stained with anti-CLIMP-63 antibody (E; red) showed a reduced ER sheet signal, relative to control cells. Nuclei in blue. Scale bars: 10 μm. (F) TEM of the ER and nuclear envelope of control (Fa) and TMEM170A-silenced cells (Fb–e). TMEM170A silencing induces nuclear envelope invagination (white arrowheads, b) and increased tubular ER (red arrowheads, Fc; another example at higher magnification in Fd). Connections between nuclear envelope and the membranes were visible (Fc, light blue arrowhead). Occasionally, ER tubules were seen connected with part of semi-organized smooth ER (cisternae, purple arrowheads) and rarely whorls (white stars) (Fe). Scale bars: 1 μm (black) or 2 μm (white). Section thickness: 60 nm.

    Article Snippet: Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analysis Ten square 25-cm dishes (each 500 cm2 ) of HeLa K TMEM170A–GFP or TMEM147–GFP cell lines or HeLa K GFP cell line (negative control) were lysed in 2 ml lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% v/v NP40 and 1 tablet/50 ml of Complete protease inhibitor cocktail (Roche)].

    Techniques: Immunofluorescence, Staining, Transmission Electron Microscopy

    Overexpression of TMEM170A induces ER sheet proliferation. (A) Immunofluorescence of untransfected HeLa K cells or transiently transfected with FLAG–TMEM170A (green) in double labeling with ER sheet marker CLIMP-63 (red); nuclei (blue). Scale bars: 10 μm. (B) TEM images of HeLa K cells untransfected (Ba) and overexpressing FLAG–TMEM170A (Bb–d). Overexpressing cells have a highly enriched ER, composed of prominent well-organized and extensive ER sheet stacks with membrane-bound ribosomes (Bb–d) as compared with untransfected cells (Ba). Small images are a magnification of a representative region (boxed). Red arrowheads indicate ribosomes present on the ER sheets, white arrowheads in Bb indicate ER sheet stacks, which are extensive throughout the cell. Scale bars: 1 μm (black) or 2 μm (white). Section thickness 60 nm. In Ba,b cells were chemically fixed; in Bc,d they were high-pressure frozen.

    Journal: Journal of Cell Science

    Article Title: Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    doi: 10.1242/jcs.175273

    Figure Lengend Snippet: Overexpression of TMEM170A induces ER sheet proliferation. (A) Immunofluorescence of untransfected HeLa K cells or transiently transfected with FLAG–TMEM170A (green) in double labeling with ER sheet marker CLIMP-63 (red); nuclei (blue). Scale bars: 10 μm. (B) TEM images of HeLa K cells untransfected (Ba) and overexpressing FLAG–TMEM170A (Bb–d). Overexpressing cells have a highly enriched ER, composed of prominent well-organized and extensive ER sheet stacks with membrane-bound ribosomes (Bb–d) as compared with untransfected cells (Ba). Small images are a magnification of a representative region (boxed). Red arrowheads indicate ribosomes present on the ER sheets, white arrowheads in Bb indicate ER sheet stacks, which are extensive throughout the cell. Scale bars: 1 μm (black) or 2 μm (white). Section thickness 60 nm. In Ba,b cells were chemically fixed; in Bc,d they were high-pressure frozen.

    Article Snippet: Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analysis Ten square 25-cm dishes (each 500 cm2 ) of HeLa K TMEM170A–GFP or TMEM147–GFP cell lines or HeLa K GFP cell line (negative control) were lysed in 2 ml lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% v/v NP40 and 1 tablet/50 ml of Complete protease inhibitor cocktail (Roche)].

    Techniques: Over Expression, Immunofluorescence, Transfection, Labeling, Marker, Transmission Electron Microscopy

    TMEM170A silencing also causes depletion or mislocalization of INM proteins. (A) TMEM170A silencing affects targeting of LBR to the INM. LBR (green) is strongly mislocalized to the ER in TMEM170A-silenced cells, compared with control cells. Triple staining with mAb414 (red) and nuclei in blue. Scale bars: 10 μm. (B) LBR (green) also localizes in aggregates in the ER (arrowheads) in most of the silenced cells, unlike in control cells. Silenced cells, stained with anti-LAP2β antibody (red), show reduced signal at the nuclear rim. Nuclei in blue. Scale bars: 10 μm. (C) Emerin (green) nuclear rim staining was reduced in TMEM170A-silenced cells, compared with controls. LAP2β (red), as in B, shows a reduced nuclear rim signal in silenced cells. Nuclei in blue. Scale bars: 10 μm. (D) LBR (red) colocalizes with calnexin aggregates (green) in the ER of TMEM170A-silenced cells. Scale bars: 10 μm. (E) Total HeLa K cell extracts from control and TMEM170A-silenced cells were assayed by western blot. The levels of LBR, emerin and calnexin were unaffected but those of LAP2β were specifically reduced in silenced cells. Two different amounts (1/5 and 1/7 of total lysate) of each protein extract were loaded on the gel, as indicated by arrows (bottom).

    Journal: Journal of Cell Science

    Article Title: Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    doi: 10.1242/jcs.175273

    Figure Lengend Snippet: TMEM170A silencing also causes depletion or mislocalization of INM proteins. (A) TMEM170A silencing affects targeting of LBR to the INM. LBR (green) is strongly mislocalized to the ER in TMEM170A-silenced cells, compared with control cells. Triple staining with mAb414 (red) and nuclei in blue. Scale bars: 10 μm. (B) LBR (green) also localizes in aggregates in the ER (arrowheads) in most of the silenced cells, unlike in control cells. Silenced cells, stained with anti-LAP2β antibody (red), show reduced signal at the nuclear rim. Nuclei in blue. Scale bars: 10 μm. (C) Emerin (green) nuclear rim staining was reduced in TMEM170A-silenced cells, compared with controls. LAP2β (red), as in B, shows a reduced nuclear rim signal in silenced cells. Nuclei in blue. Scale bars: 10 μm. (D) LBR (red) colocalizes with calnexin aggregates (green) in the ER of TMEM170A-silenced cells. Scale bars: 10 μm. (E) Total HeLa K cell extracts from control and TMEM170A-silenced cells were assayed by western blot. The levels of LBR, emerin and calnexin were unaffected but those of LAP2β were specifically reduced in silenced cells. Two different amounts (1/5 and 1/7 of total lysate) of each protein extract were loaded on the gel, as indicated by arrows (bottom).

    Article Snippet: Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analysis Ten square 25-cm dishes (each 500 cm2 ) of HeLa K TMEM170A–GFP or TMEM147–GFP cell lines or HeLa K GFP cell line (negative control) were lysed in 2 ml lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% v/v NP40 and 1 tablet/50 ml of Complete protease inhibitor cocktail (Roche)].

    Techniques: Staining, Western Blot

    Downregulation of TMEM170A by siRNA alters nuclear shape and size and causes a significant decrease in NPC numbers. (A) siRNA knockdown of TMEM170A reduced nuclear rim signal in HeLa K cells. Cells were transfected with negative control or TMEM170A siRNAs and, at 72 h post-transfection, fixed and stained with mAb414 (green), anti-MEL28/ELYS (red) or anti-Pom121 (white) antibodies. Nuclei counterstained in blue in all panels. Scale bars: 10 μm. (B) TMEM170A siRNA also causes reduction of nuclear rim signal in U2OS cells. Control or TMEM170A-silenced U2OS cells were stained with mAb414 (red) and anti-MEL28/ELYS (green) or anti-Pom121 (white) antibodies. Scale bars: 10 μm (left panels) or 5 μm (right panels). (C) NPCs, at the bottom surface of the nuclear envelope of control- and TMEM170A-silenced HeLa K cells, were visualized by indirect immunofluorescence using mAb414 antibody. Representative areas of each cell are magnified in the boxes. Scale bars: 5 μm. (D) Quantitation of mean immunofluorescence of anti-MEL28/ELYS and mAb414 antibodies to estimate NPC density in HeLa K cells treated with control or TMEM170A siRNAs ( n > 52 nuclei per condition from three independent experiments). Error bars show s.d.; * P

    Journal: Journal of Cell Science

    Article Title: Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    doi: 10.1242/jcs.175273

    Figure Lengend Snippet: Downregulation of TMEM170A by siRNA alters nuclear shape and size and causes a significant decrease in NPC numbers. (A) siRNA knockdown of TMEM170A reduced nuclear rim signal in HeLa K cells. Cells were transfected with negative control or TMEM170A siRNAs and, at 72 h post-transfection, fixed and stained with mAb414 (green), anti-MEL28/ELYS (red) or anti-Pom121 (white) antibodies. Nuclei counterstained in blue in all panels. Scale bars: 10 μm. (B) TMEM170A siRNA also causes reduction of nuclear rim signal in U2OS cells. Control or TMEM170A-silenced U2OS cells were stained with mAb414 (red) and anti-MEL28/ELYS (green) or anti-Pom121 (white) antibodies. Scale bars: 10 μm (left panels) or 5 μm (right panels). (C) NPCs, at the bottom surface of the nuclear envelope of control- and TMEM170A-silenced HeLa K cells, were visualized by indirect immunofluorescence using mAb414 antibody. Representative areas of each cell are magnified in the boxes. Scale bars: 5 μm. (D) Quantitation of mean immunofluorescence of anti-MEL28/ELYS and mAb414 antibodies to estimate NPC density in HeLa K cells treated with control or TMEM170A siRNAs ( n > 52 nuclei per condition from three independent experiments). Error bars show s.d.; * P

    Article Snippet: Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analysis Ten square 25-cm dishes (each 500 cm2 ) of HeLa K TMEM170A–GFP or TMEM147–GFP cell lines or HeLa K GFP cell line (negative control) were lysed in 2 ml lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% v/v NP40 and 1 tablet/50 ml of Complete protease inhibitor cocktail (Roche)].

    Techniques: Transfection, Negative Control, Staining, Immunofluorescence, Quantitation Assay

    Localization and membrane topology of human TMEM170A. (A) TMEM170A localizes to the nuclear envelope and the ER. (Aa–c) Immunofluorescence of HeLa K cells transiently transfected with TMEM170A–GFP (green), FLAG–TMEM170A (green) or myc–TMEM170A (red). DNA was visualized with Hoechst in all panels (blue). (Ad) Immunofluorescence of HeLa K cells transiently transfected with TMEM170A–GFP (green) and co-stained for ER markers calnexin (red) or RTN4/NogoA+B (red). (B) The C-terminal domain of TMEM170A faces the cytoplasm. HeLa K cells, stably expressing TMEM170A–GFP, were fixed and subjected to 0.05% w/v digitonin to permeabilize the plasma membrane or 0.05% w/v digitonin+0.5% v/v Triton X-100 to permeabilize both the plasma membrane and the nuclear envelope. Cells were stained with anti-GFP antibody (green) in combination with either anti-LAP2β or anti-lamin-A or calreticulin (red). TMEM170A–GFP is accessible to the anti-GFP antibody in digitonin-only semi-permeabilized cells, whereas LAP2β, lamin A and calreticulin are only recognized upon full permeabilization. (C) Schematic of the membrane topology of TMEM170A. Transmembrane segments were predicted with TMPRED and topology of the C-terminus was confirmed in the experiments shown in B that support the model shown. Scale bars: 10 μm.

    Journal: Journal of Cell Science

    Article Title: Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    doi: 10.1242/jcs.175273

    Figure Lengend Snippet: Localization and membrane topology of human TMEM170A. (A) TMEM170A localizes to the nuclear envelope and the ER. (Aa–c) Immunofluorescence of HeLa K cells transiently transfected with TMEM170A–GFP (green), FLAG–TMEM170A (green) or myc–TMEM170A (red). DNA was visualized with Hoechst in all panels (blue). (Ad) Immunofluorescence of HeLa K cells transiently transfected with TMEM170A–GFP (green) and co-stained for ER markers calnexin (red) or RTN4/NogoA+B (red). (B) The C-terminal domain of TMEM170A faces the cytoplasm. HeLa K cells, stably expressing TMEM170A–GFP, were fixed and subjected to 0.05% w/v digitonin to permeabilize the plasma membrane or 0.05% w/v digitonin+0.5% v/v Triton X-100 to permeabilize both the plasma membrane and the nuclear envelope. Cells were stained with anti-GFP antibody (green) in combination with either anti-LAP2β or anti-lamin-A or calreticulin (red). TMEM170A–GFP is accessible to the anti-GFP antibody in digitonin-only semi-permeabilized cells, whereas LAP2β, lamin A and calreticulin are only recognized upon full permeabilization. (C) Schematic of the membrane topology of TMEM170A. Transmembrane segments were predicted with TMPRED and topology of the C-terminus was confirmed in the experiments shown in B that support the model shown. Scale bars: 10 μm.

    Article Snippet: Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analysis Ten square 25-cm dishes (each 500 cm2 ) of HeLa K TMEM170A–GFP or TMEM147–GFP cell lines or HeLa K GFP cell line (negative control) were lysed in 2 ml lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% v/v NP40 and 1 tablet/50 ml of Complete protease inhibitor cocktail (Roche)].

    Techniques: Immunofluorescence, Transfection, Staining, Stable Transfection, Expressing

    FLAG–TMEM170A expression rescues the phenotype caused by TMEM170A silencing in HeLa K cells. (A) HeLa K cells were transfected with control or TMEM170A siRNAs and, 48 h later, with a plasmid expressing FLAG–TMEM170A. Cells were fixed after a further 24 h and stained with anti-MEL28/ELYS (red) and anti-FLAG (green) antibodies. Nuclei in blue. Scale bars: 10 μm. (B) Quantitation of nuclear size (Ba), nuclear volume (Bb), and mean nuclear immunofluorescence of anti-MEL28/ELYS antibody signal to estimate the NPC density (Bc) of cells silenced with control or TMEM170A siRNAi and transiently transfected or not with FLAG–TMEM170A [HeLa K+ control RNAi ( n =52 cells), HeLa K+ TMEM170A RNAi ( n =52), FLAG–TMEM170A+control RNAi ( n =52) and FLAG–TMEM170A+TMEM170A RNAi ( n =34)]. For each parameter, the average of three independent experiments per condition is displayed; error bars show s.d.; * P

    Journal: Journal of Cell Science

    Article Title: Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    doi: 10.1242/jcs.175273

    Figure Lengend Snippet: FLAG–TMEM170A expression rescues the phenotype caused by TMEM170A silencing in HeLa K cells. (A) HeLa K cells were transfected with control or TMEM170A siRNAs and, 48 h later, with a plasmid expressing FLAG–TMEM170A. Cells were fixed after a further 24 h and stained with anti-MEL28/ELYS (red) and anti-FLAG (green) antibodies. Nuclei in blue. Scale bars: 10 μm. (B) Quantitation of nuclear size (Ba), nuclear volume (Bb), and mean nuclear immunofluorescence of anti-MEL28/ELYS antibody signal to estimate the NPC density (Bc) of cells silenced with control or TMEM170A siRNAi and transiently transfected or not with FLAG–TMEM170A [HeLa K+ control RNAi ( n =52 cells), HeLa K+ TMEM170A RNAi ( n =52), FLAG–TMEM170A+control RNAi ( n =52) and FLAG–TMEM170A+TMEM170A RNAi ( n =34)]. For each parameter, the average of three independent experiments per condition is displayed; error bars show s.d.; * P

    Article Snippet: Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analysis Ten square 25-cm dishes (each 500 cm2 ) of HeLa K TMEM170A–GFP or TMEM147–GFP cell lines or HeLa K GFP cell line (negative control) were lysed in 2 ml lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% v/v NP40 and 1 tablet/50 ml of Complete protease inhibitor cocktail (Roche)].

    Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Quantitation Assay, Immunofluorescence

    TMEM170A interacts with RTN4. (A) Silver-stained SDS-PAGE gel showing GFP (line 1), GFP–TMEM147 (line 2) control IPs and TMEM170A–GFP IP (line 3) with the use of GFP-Trap_A beads. Three bands, uniquely present in TMEM170A–GFP IP (arrows), were isolated and identified by liquid chromatography coupled with tandem mass spectrometry ( Table S1 ). Band 2 (circled in red) was RTN4. (B) The anti-GFP IP was repeated and the interaction between TMEM170A and RTN4 was confirmed by western blot, using anti-GFP and anti-RTN4/NogoA+B antibodies. ‘Input’ corresponds to 1/40 volume of the lysate used for the reaction and ‘IP’ is 1/2 of the bound fraction, i.e. the protein complexes captured on the beads.

    Journal: Journal of Cell Science

    Article Title: Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    doi: 10.1242/jcs.175273

    Figure Lengend Snippet: TMEM170A interacts with RTN4. (A) Silver-stained SDS-PAGE gel showing GFP (line 1), GFP–TMEM147 (line 2) control IPs and TMEM170A–GFP IP (line 3) with the use of GFP-Trap_A beads. Three bands, uniquely present in TMEM170A–GFP IP (arrows), were isolated and identified by liquid chromatography coupled with tandem mass spectrometry ( Table S1 ). Band 2 (circled in red) was RTN4. (B) The anti-GFP IP was repeated and the interaction between TMEM170A and RTN4 was confirmed by western blot, using anti-GFP and anti-RTN4/NogoA+B antibodies. ‘Input’ corresponds to 1/40 volume of the lysate used for the reaction and ‘IP’ is 1/2 of the bound fraction, i.e. the protein complexes captured on the beads.

    Article Snippet: Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analysis Ten square 25-cm dishes (each 500 cm2 ) of HeLa K TMEM170A–GFP or TMEM147–GFP cell lines or HeLa K GFP cell line (negative control) were lysed in 2 ml lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% v/v NP40 and 1 tablet/50 ml of Complete protease inhibitor cocktail (Roche)].

    Techniques: Staining, SDS Page, Isolation, Liquid Chromatography, Mass Spectrometry, Western Blot

    Double TMEM170A plus RTN4 silencing restores the phenotypes caused either by single TMEM170A- or RTN4-silencing in HeLa K cells. (A,B) Comparison of ER structure in cells silenced with control, single TMEM170A, RTN4 and double TMEM170A plus RTN4 RNAi, stained with anti-calnexin or anti-RTN4 (green) and mAb414 (red) showing that double silencing mostly reverses aberrant ER morphology and reduced nuclear rim signal induced by single TMEM170A silencing. Double TMEM170A- plus RTN4-silenced cells showed no altered phenotype, resembling control cells (upper row). (C) Equivalent experiment as in A,B, with cells stained for LAP2β (red) and emerin (green) or LBR (white). Single TMEM170A-silenced cells typically displayed reduced nuclear rim LAP2β or emerin signal and LBR was mislocalized to the ER. Single RTN4-silenced cells showed no phenotype but the double TMEM170A- plus RTN4-silenced cells exhibited restoration of LAP2β, emerin and LBR proteins to the nuclear envelope rim, as in controls. Nuclei in blue. Scale bars: 10 μm. (D) Western blot analysis of samples silenced with control, single TMEM170A, RTN4, or double TMEM170A plus RTN4 RNAi. Simultaneous TMEM170A plus RTN4 silencing restored to some extent the protein levels of nucleoporin Nup62 and LAP2β, compared with single TMEM170A or RTN4 silencing (Da). Single TMEM170A or RTN4 silencing and double TMEM170A plus RTN4 silencing have no effect on calnexin and emerin protein levels (Db).

    Journal: Journal of Cell Science

    Article Title: Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    doi: 10.1242/jcs.175273

    Figure Lengend Snippet: Double TMEM170A plus RTN4 silencing restores the phenotypes caused either by single TMEM170A- or RTN4-silencing in HeLa K cells. (A,B) Comparison of ER structure in cells silenced with control, single TMEM170A, RTN4 and double TMEM170A plus RTN4 RNAi, stained with anti-calnexin or anti-RTN4 (green) and mAb414 (red) showing that double silencing mostly reverses aberrant ER morphology and reduced nuclear rim signal induced by single TMEM170A silencing. Double TMEM170A- plus RTN4-silenced cells showed no altered phenotype, resembling control cells (upper row). (C) Equivalent experiment as in A,B, with cells stained for LAP2β (red) and emerin (green) or LBR (white). Single TMEM170A-silenced cells typically displayed reduced nuclear rim LAP2β or emerin signal and LBR was mislocalized to the ER. Single RTN4-silenced cells showed no phenotype but the double TMEM170A- plus RTN4-silenced cells exhibited restoration of LAP2β, emerin and LBR proteins to the nuclear envelope rim, as in controls. Nuclei in blue. Scale bars: 10 μm. (D) Western blot analysis of samples silenced with control, single TMEM170A, RTN4, or double TMEM170A plus RTN4 RNAi. Simultaneous TMEM170A plus RTN4 silencing restored to some extent the protein levels of nucleoporin Nup62 and LAP2β, compared with single TMEM170A or RTN4 silencing (Da). Single TMEM170A or RTN4 silencing and double TMEM170A plus RTN4 silencing have no effect on calnexin and emerin protein levels (Db).

    Article Snippet: Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analysis Ten square 25-cm dishes (each 500 cm2 ) of HeLa K TMEM170A–GFP or TMEM147–GFP cell lines or HeLa K GFP cell line (negative control) were lysed in 2 ml lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% v/v NP40 and 1 tablet/50 ml of Complete protease inhibitor cocktail (Roche)].

    Techniques: Staining, Western Blot

    RCA can detect rSDS-PrP Sc oligomers at early stages of the disease. a Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [ 16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels. b Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above. c To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of proteinase K and processed as described in the legend to Fig. 4a . The asterisk in b and c indicates the position of oligomer traces

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: RCA can detect rSDS-PrP Sc oligomers at early stages of the disease. a Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [ 16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels. b Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above. c To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of proteinase K and processed as described in the legend to Fig. 4a . The asterisk in b and c indicates the position of oligomer traces

    Article Snippet: Biological reagents and antibodies Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Infection, Incubation, Western Blot

    A MR100-based assay can differentiate between prion-infected and normal brain homogenates without proteinase K digestion. a Schematic description of the RCA protocol to test brain homogenates without PK digestion. Brain tissues were freshly homogenized in microbead-containing tubes. Normal brain homogenates (NBH) or prion-infected brain homogenates (IBH) were incubated with MR100 for 1 h, at room temperature, leading to a precipitation of PrP isoforms. After a short centrifugation step, the pellet with MR100 (orange tube) concentrates PrP isoforms, whereas no pellet is detectable with DMSO. b Comparison of DMSO, P30 and MR100 precipitation capabilities using the RCA protocol. Fifty μL of 10 % 22 L infected brain homogenates were diluted in 300 μL of PBS/2 % sarcosyl and incubated using either 1.5 mM of P30 or MR100 or an equivalent volume of the solvent alone (DMSO), at room temperature for 1 h. Then, samples were centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant was mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with an equal volume of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix according to standard procedures [ 16 ]. The samples (S/P) were analyzed by western blotting using SAF mix anti-PrP antibodies. c Comparison of infected versus non-infected brain homogenates processed with the RCA protocol. Fifty microliters of 10 % freshly homogenized brain tissues from normal (NBH) or 22 L prion-infected (IBH) mice were processed according to the RCA protocol described in A and B. Thirty microliters of supernatant (S) or pellet (P) were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix as described above. Molecular masses (20–75 kDa) are indicated on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: A MR100-based assay can differentiate between prion-infected and normal brain homogenates without proteinase K digestion. a Schematic description of the RCA protocol to test brain homogenates without PK digestion. Brain tissues were freshly homogenized in microbead-containing tubes. Normal brain homogenates (NBH) or prion-infected brain homogenates (IBH) were incubated with MR100 for 1 h, at room temperature, leading to a precipitation of PrP isoforms. After a short centrifugation step, the pellet with MR100 (orange tube) concentrates PrP isoforms, whereas no pellet is detectable with DMSO. b Comparison of DMSO, P30 and MR100 precipitation capabilities using the RCA protocol. Fifty μL of 10 % 22 L infected brain homogenates were diluted in 300 μL of PBS/2 % sarcosyl and incubated using either 1.5 mM of P30 or MR100 or an equivalent volume of the solvent alone (DMSO), at room temperature for 1 h. Then, samples were centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant was mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with an equal volume of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix according to standard procedures [ 16 ]. The samples (S/P) were analyzed by western blotting using SAF mix anti-PrP antibodies. c Comparison of infected versus non-infected brain homogenates processed with the RCA protocol. Fifty microliters of 10 % freshly homogenized brain tissues from normal (NBH) or 22 L prion-infected (IBH) mice were processed according to the RCA protocol described in A and B. Thirty microliters of supernatant (S) or pellet (P) were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix as described above. Molecular masses (20–75 kDa) are indicated on the left side of the panels

    Article Snippet: Biological reagents and antibodies Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Infection, Incubation, Centrifugation, Western Blot, Mouse Assay

    rSDS-PrP Sc oligomers are detected in patients with new variant CJD (vCJD) when tested with RCA. a-b Frozen, homogenized brain samples from two patients with vCJD, one patient with sCJD (codon 129 M/M genotype) (positive control) and one healthy control (NBH) were from NIBSC. Each sample was identified by the number attributed by the NIBSC. The RCA assay was carried out as before (see legend to Fig. 5 ) but adapted to human samples: 50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl were incubated with 2 mM MR100 for 2 h. Before centrifugation, 30 μL was collected and mixed with 30 μL of 2X loading buffer for immunoblotting analysis ( a ). The rest of the sample was centrifuged at 11,000 g for 5 min, and supernatants (S) and pellets (P) were immunoblotted with the SAFmix ( b ) [ 16 ]. c-d For comparison, the same brain homogenates (50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl) were processed with the classical proteinase K digestion assay without MR100. Samples were analyzed before ( c ), and after proteinase K digestion (125 μg/mL PK at 37 °C for 1 h) ( d ). The reaction was stopped by addition of a protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix. Molecular masses (20–75 kDa) are on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: rSDS-PrP Sc oligomers are detected in patients with new variant CJD (vCJD) when tested with RCA. a-b Frozen, homogenized brain samples from two patients with vCJD, one patient with sCJD (codon 129 M/M genotype) (positive control) and one healthy control (NBH) were from NIBSC. Each sample was identified by the number attributed by the NIBSC. The RCA assay was carried out as before (see legend to Fig. 5 ) but adapted to human samples: 50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl were incubated with 2 mM MR100 for 2 h. Before centrifugation, 30 μL was collected and mixed with 30 μL of 2X loading buffer for immunoblotting analysis ( a ). The rest of the sample was centrifuged at 11,000 g for 5 min, and supernatants (S) and pellets (P) were immunoblotted with the SAFmix ( b ) [ 16 ]. c-d For comparison, the same brain homogenates (50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl) were processed with the classical proteinase K digestion assay without MR100. Samples were analyzed before ( c ), and after proteinase K digestion (125 μg/mL PK at 37 °C for 1 h) ( d ). The reaction was stopped by addition of a protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix. Molecular masses (20–75 kDa) are on the left side of the panels

    Article Snippet: Biological reagents and antibodies Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Variant Assay, Positive Control, Incubation, Centrifugation, Protease Inhibitor, Western Blot

    MR100 has a stronger PrP Sc oligomer-inducing activity in prion-infected N2a58/22 L cells than P30 and A6. a Effect of the newly synthesized MR1, MR2 and MR100 compounds in prion-infected N2a58/22 L cells. Cells were left untreated (CTR) or incubated with 20 μM of A6 (positive control), MR1 and MR2 (synthesis intermediates), MR100, or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix (a mixture of the anti-PrP SAF60, SAF69 and SAF70 monoclonal antibodies) after proteinase K (PK) digestion. b Comparison of the oligomer-inducing activity of P30, A6 and MR100. Prion-infected N2a58/22 L cells were incubated with 0.5, 1 or 2.5 μM of each compound, or 20 μL DMSO (DM) for 4 days. Protein lysates were then analyzed by immunoblotting with the SAF mix after PK digestion. c MR100 dose-response curve in prion-infected N2a58/22 L cells. Successive dilutions of MR100 in DMSO were used to obtain final concentrations ranging from 10 -12 M (1pM) to 10 -5 M (10 μM). Cells were incubated for 4 days and at confluence they were lysed. Protein lysates were analyzed by immunoblotting with the SAF mix after PK digestion according to the previously described protocol [ 16 , 30 ]. Loading control was performed with antibodies against glyceraldehyde-3-P dehydrogenase (G3PDH) and before proteinase K digestion. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: MR100 has a stronger PrP Sc oligomer-inducing activity in prion-infected N2a58/22 L cells than P30 and A6. a Effect of the newly synthesized MR1, MR2 and MR100 compounds in prion-infected N2a58/22 L cells. Cells were left untreated (CTR) or incubated with 20 μM of A6 (positive control), MR1 and MR2 (synthesis intermediates), MR100, or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix (a mixture of the anti-PrP SAF60, SAF69 and SAF70 monoclonal antibodies) after proteinase K (PK) digestion. b Comparison of the oligomer-inducing activity of P30, A6 and MR100. Prion-infected N2a58/22 L cells were incubated with 0.5, 1 or 2.5 μM of each compound, or 20 μL DMSO (DM) for 4 days. Protein lysates were then analyzed by immunoblotting with the SAF mix after PK digestion. c MR100 dose-response curve in prion-infected N2a58/22 L cells. Successive dilutions of MR100 in DMSO were used to obtain final concentrations ranging from 10 -12 M (1pM) to 10 -5 M (10 μM). Cells were incubated for 4 days and at confluence they were lysed. Protein lysates were analyzed by immunoblotting with the SAF mix after PK digestion according to the previously described protocol [ 16 , 30 ]. Loading control was performed with antibodies against glyceraldehyde-3-P dehydrogenase (G3PDH) and before proteinase K digestion. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Article Snippet: Biological reagents and antibodies Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Activity Assay, Infection, Synthesized, Incubation, Positive Control

    MR100 did not induce SDS resistant PrP C oligomers. a Parental non-infected cells, N2a58, were left untreated (CTR) or incubated with 20 μM of MR100 or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix before or after PK digestion. b N2a58 cell lines were left untreated (CTR) or incubated with various concentration of MR100 from 5 to 40 μM or 40 μL of DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF 32 before or after PK digestion. Loading control was performed with antibodies against β actin and before proteinase K digestion. c Schematic representation of the protocols used to test if PrP C isoforms are part of rSDS-oligomers (Left panel). First step, N2a58/22 L lysates were incubated with 20 μM of MR100 or with proteinase K to eliminate PrP C , then in the second step, MR100-exposed lysates were digested with proteinase K, while proteinase K digested samples were incubated with MR100. PrP Sc species were then analyzed by western blotting. Western blot analysis of the samples processed according to the two different protocols using the SAFmix of anti-PrP antibodies (Right panel). CTR, untreated samples, digested by proteinase K; DM, DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: MR100 did not induce SDS resistant PrP C oligomers. a Parental non-infected cells, N2a58, were left untreated (CTR) or incubated with 20 μM of MR100 or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix before or after PK digestion. b N2a58 cell lines were left untreated (CTR) or incubated with various concentration of MR100 from 5 to 40 μM or 40 μL of DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF 32 before or after PK digestion. Loading control was performed with antibodies against β actin and before proteinase K digestion. c Schematic representation of the protocols used to test if PrP C isoforms are part of rSDS-oligomers (Left panel). First step, N2a58/22 L lysates were incubated with 20 μM of MR100 or with proteinase K to eliminate PrP C , then in the second step, MR100-exposed lysates were digested with proteinase K, while proteinase K digested samples were incubated with MR100. PrP Sc species were then analyzed by western blotting. Western blot analysis of the samples processed according to the two different protocols using the SAFmix of anti-PrP antibodies (Right panel). CTR, untreated samples, digested by proteinase K; DM, DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Article Snippet: Biological reagents and antibodies Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Infection, Incubation, Concentration Assay, Western Blot

    MR100 shows oligomer-inducing activity in brain homogenates from prion-infected rodents. a MR100 oligomer-inducing activity was tested using freshly homogenized rodent brain tissues infected with the 22 L (mice) or the 263 K prion strain (hamsters). Fifty μL of 10 % mouse or hamster brain homogenates were diluted in 300 μL PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 (corresponding to 150 μL of 5 mM MR100) at room temperature for 1 h or with 150 μL of DMSO as control, and then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins). PK digestion was stopped by addition of a cocktail of protease inhibitors (Complete), before analysis of rPrP Sc by western blotting with the SAF mix according to the previously described protocol [ 16 ]. CTR: untreated 22 L- or 263 K-infected brain homogenates (negative control); DM: 22 L- or 263 K-infected brain homogenates incubated with DMSO. b Comparison of P30 and MR100 oligomer-inducing activity on the 22 L prion strain, before and after proteinase K digestion. Fifty μL of 10 % 22 L-infected brain homogenates were diluted in 350 μL PBS/2 % Sarkosyl, incubated with 1 mM MR100 or P30 (corresponding to 100 μL of 5 mM MR100 or P30), at room temperature for 1 h. Then, aliquots of 30 μL were taken before addition of proteinase K, to perform western blot (PK-) probed with SAF mix, but also with anti-β-actin antibodies as loading controls. The rest of the sample was then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins) (PK+). The reaction was stopped by addition of the protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix as in A. DM: 22 L-infected brain homogenates incubated with 100 μL DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: MR100 shows oligomer-inducing activity in brain homogenates from prion-infected rodents. a MR100 oligomer-inducing activity was tested using freshly homogenized rodent brain tissues infected with the 22 L (mice) or the 263 K prion strain (hamsters). Fifty μL of 10 % mouse or hamster brain homogenates were diluted in 300 μL PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 (corresponding to 150 μL of 5 mM MR100) at room temperature for 1 h or with 150 μL of DMSO as control, and then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins). PK digestion was stopped by addition of a cocktail of protease inhibitors (Complete), before analysis of rPrP Sc by western blotting with the SAF mix according to the previously described protocol [ 16 ]. CTR: untreated 22 L- or 263 K-infected brain homogenates (negative control); DM: 22 L- or 263 K-infected brain homogenates incubated with DMSO. b Comparison of P30 and MR100 oligomer-inducing activity on the 22 L prion strain, before and after proteinase K digestion. Fifty μL of 10 % 22 L-infected brain homogenates were diluted in 350 μL PBS/2 % Sarkosyl, incubated with 1 mM MR100 or P30 (corresponding to 100 μL of 5 mM MR100 or P30), at room temperature for 1 h. Then, aliquots of 30 μL were taken before addition of proteinase K, to perform western blot (PK-) probed with SAF mix, but also with anti-β-actin antibodies as loading controls. The rest of the sample was then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins) (PK+). The reaction was stopped by addition of the protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix as in A. DM: 22 L-infected brain homogenates incubated with 100 μL DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Article Snippet: Biological reagents and antibodies Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Activity Assay, Infection, Mouse Assay, Incubation, Western Blot, Negative Control, Protease Inhibitor

    Glucose starvation promotes efficient polarized cell growth and induces the expression of mating-related genes in MTL a/a cells of C . albicans . (A) Morphologies of the laboratory strain GH1013 ( MTL a / a ) grown on YPD-K and YP-K media. 1 × 10 5 cells were spotted on YPD-K and YP-K media and cultured at 25°C for five days. Scale bar for colonies (left panels), 2 mm; scale bar for cells (right panels), 10 μm. (B) Morphologies of three clinical C . albicans strains ( MTL a / a ) grown on YP-K medium at 30°C for four days. Scale bar for colonies (inset), 2 mm; scale bar for cells, 10 μm. (C) Relative expression levels of mating-related genes normalized to ACT1 . Cells of GH1013 were used, and culture conditions were same as described in panel (A). Error bars represent standard errors of technical duplicates. * p

    Journal: PLoS Biology

    Article Title: Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1–Hsp90 pathway

    doi: 10.1371/journal.pbio.2006966

    Figure Lengend Snippet: Glucose starvation promotes efficient polarized cell growth and induces the expression of mating-related genes in MTL a/a cells of C . albicans . (A) Morphologies of the laboratory strain GH1013 ( MTL a / a ) grown on YPD-K and YP-K media. 1 × 10 5 cells were spotted on YPD-K and YP-K media and cultured at 25°C for five days. Scale bar for colonies (left panels), 2 mm; scale bar for cells (right panels), 10 μm. (B) Morphologies of three clinical C . albicans strains ( MTL a / a ) grown on YP-K medium at 30°C for four days. Scale bar for colonies (inset), 2 mm; scale bar for cells, 10 μm. (C) Relative expression levels of mating-related genes normalized to ACT1 . Cells of GH1013 were used, and culture conditions were same as described in panel (A). Error bars represent standard errors of technical duplicates. * p

    Article Snippet: Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany).

    Techniques: Expressing, Cell Culture

    Down-regulation of Hsp90 promotes the development of mating projections. (A) Morphologies of the control and tetON-HSP90/hsp90 mutant. Control, GH1013 + p ACT1 - WOR1 ; tetON-HSP90/hsp90 , a tetON -promoter–controlled conditional expression strain of HSP90 with ectopically expressed WOR1 (+ pACT1 - WOR1 ). 1 × 10 5 cells were spotted on YPD-K and YP-K media with or without Dox as indicated and cultured at 25°C for three, five, or seven days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (B) Relative expression of mating-related genes. 1 × 10 5 cells of each strain were cultured on YPD-K medium (for seven days) or on YP-K medium (for three days) with 40 μg/mL Dox at 25°C. Relative expression levels were not tested on YP-K medium without Dox since cell viability of the tetON-HSP90/hsp90 mutant was severely impaired. Error bars represent standard errors of technical duplicates. * p

    Journal: PLoS Biology

    Article Title: Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1–Hsp90 pathway

    doi: 10.1371/journal.pbio.2006966

    Figure Lengend Snippet: Down-regulation of Hsp90 promotes the development of mating projections. (A) Morphologies of the control and tetON-HSP90/hsp90 mutant. Control, GH1013 + p ACT1 - WOR1 ; tetON-HSP90/hsp90 , a tetON -promoter–controlled conditional expression strain of HSP90 with ectopically expressed WOR1 (+ pACT1 - WOR1 ). 1 × 10 5 cells were spotted on YPD-K and YP-K media with or without Dox as indicated and cultured at 25°C for three, five, or seven days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (B) Relative expression of mating-related genes. 1 × 10 5 cells of each strain were cultured on YPD-K medium (for seven days) or on YP-K medium (for three days) with 40 μg/mL Dox at 25°C. Relative expression levels were not tested on YP-K medium without Dox since cell viability of the tetON-HSP90/hsp90 mutant was severely impaired. Error bars represent standard errors of technical duplicates. * p

    Article Snippet: Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany).

    Techniques: Mutagenesis, Expressing, Cell Culture

    Role of Hsf1 in the induction of mating projections in C . albicans . (A) Morphologies of the control and tetON-HSF1/hsf1 mutant. 1 × 10 5 cells were spotted on YPD-K and YP-K media without or with 40 μg/mL Dox and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. Control, GH1013cartTA. (B) Relative expression levels of mating-related genes. 1 × 10 5 cells of each strain were cultured on YPD-K medium (for six days) or on YP-K medium (for three days) without or with 40 μg/mL Dox at 25°C. Error bars represent standard errors of technical duplicates. * p

    Journal: PLoS Biology

    Article Title: Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1–Hsp90 pathway

    doi: 10.1371/journal.pbio.2006966

    Figure Lengend Snippet: Role of Hsf1 in the induction of mating projections in C . albicans . (A) Morphologies of the control and tetON-HSF1/hsf1 mutant. 1 × 10 5 cells were spotted on YPD-K and YP-K media without or with 40 μg/mL Dox and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. Control, GH1013cartTA. (B) Relative expression levels of mating-related genes. 1 × 10 5 cells of each strain were cultured on YPD-K medium (for six days) or on YP-K medium (for three days) without or with 40 μg/mL Dox at 25°C. Error bars represent standard errors of technical duplicates. * p

    Article Snippet: Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany).

    Techniques: Mutagenesis, Cell Culture, Expressing

    Glucose starvation induces same-sex mating in C . albicans . (A) Same-sex mating between two “ a ” strains (GH1013 and GH1350a). 1 × 10 7 cells of each strain were mixed and cultured on YPD-K and YP-K media at 25°C. After three days of growth, the mating mixture was replated onto SCD-Arg, SCD-His, and SCD-Arg-His dropout plates to assess mating efficiency. The middle panel (up) indicates that two “ a ” cells underwent cell fusion and a daughter cell grew out from the conjunction tube. Scale bar, 10 μm. (B) PCR verification of the MTL types. Strains used: lane 1, SN152 ( a /α); 2, GH1350 (α/α); 3, GH1350a ( a / a ); 4, GH1013 ( a / a ); 5–7, progeny strains of the GH1350a × GH1013a cross. (C) FACS analysis of the DNA content of parental and progeny strains. Parental diploids have the standard G1 and G2 cell cycle peaks representing 2C and 4C DNA levels. Mating progeny contain DNA content corresponding to 4C and 8C peaks, confirming their tetraploid nature. Arg, arginine; d, daughter cell; FACS, fluorescence-activated cell sorting; His, histidine; M, DNA ladder; MTL , Mating type locus; p, mating projection; SCD, synthetic complete medium; YPD-K, yeast extract-peptone-glucose-K 2 HPO 4 ; YP-K, yeast extract-peptone-K 2 HPO 4 .

    Journal: PLoS Biology

    Article Title: Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1–Hsp90 pathway

    doi: 10.1371/journal.pbio.2006966

    Figure Lengend Snippet: Glucose starvation induces same-sex mating in C . albicans . (A) Same-sex mating between two “ a ” strains (GH1013 and GH1350a). 1 × 10 7 cells of each strain were mixed and cultured on YPD-K and YP-K media at 25°C. After three days of growth, the mating mixture was replated onto SCD-Arg, SCD-His, and SCD-Arg-His dropout plates to assess mating efficiency. The middle panel (up) indicates that two “ a ” cells underwent cell fusion and a daughter cell grew out from the conjunction tube. Scale bar, 10 μm. (B) PCR verification of the MTL types. Strains used: lane 1, SN152 ( a /α); 2, GH1350 (α/α); 3, GH1350a ( a / a ); 4, GH1013 ( a / a ); 5–7, progeny strains of the GH1350a × GH1013a cross. (C) FACS analysis of the DNA content of parental and progeny strains. Parental diploids have the standard G1 and G2 cell cycle peaks representing 2C and 4C DNA levels. Mating progeny contain DNA content corresponding to 4C and 8C peaks, confirming their tetraploid nature. Arg, arginine; d, daughter cell; FACS, fluorescence-activated cell sorting; His, histidine; M, DNA ladder; MTL , Mating type locus; p, mating projection; SCD, synthetic complete medium; YPD-K, yeast extract-peptone-glucose-K 2 HPO 4 ; YP-K, yeast extract-peptone-K 2 HPO 4 .

    Article Snippet: Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany).

    Techniques: Cell Culture, Polymerase Chain Reaction, FACS, Fluorescence

    Oxidative stress promotes the development of mating projections in C . albicans . (A) Relative ROS levels. 1 × 10 5 cells of strain GH1013 were spotted on YPD-K and YP-K media and cultured at 25°C for one, two, three, or five days. For each day, 1 × 10 6 cells were used for ROS level determination. Error bars represent standard deviation of three biological replicates. * indicates significant difference ( p

    Journal: PLoS Biology

    Article Title: Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1–Hsp90 pathway

    doi: 10.1371/journal.pbio.2006966

    Figure Lengend Snippet: Oxidative stress promotes the development of mating projections in C . albicans . (A) Relative ROS levels. 1 × 10 5 cells of strain GH1013 were spotted on YPD-K and YP-K media and cultured at 25°C for one, two, three, or five days. For each day, 1 × 10 6 cells were used for ROS level determination. Error bars represent standard deviation of three biological replicates. * indicates significant difference ( p

    Article Snippet: Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany).

    Techniques: Cell Culture, Standard Deviation

    Role of the Cwt1 transcription factor in the induction of mating projections. (A and B) Morphologies of the control (GH1013 + ARG4 + HIS1 ) and cwt1/cwt1 mutant on YP-K (A) or YPD-K (B) medium. 1 × 10 5 cells of each strain were spotted on YPD-K and YP-K media and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (C) Relative expression levels of CWT1 in YPD-K and YP-K media. Cells of C . albicans were spotted on YPD-K and YP-K media and cultured at 25°C for five days. (D) Relative expression levels of CWT1 in the control (GH1013cartTA) and tetON-HSF1/hsf1 on YP-K medium. Error bars, standard errors of technical duplicates. * p

    Journal: PLoS Biology

    Article Title: Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1–Hsp90 pathway

    doi: 10.1371/journal.pbio.2006966

    Figure Lengend Snippet: Role of the Cwt1 transcription factor in the induction of mating projections. (A and B) Morphologies of the control (GH1013 + ARG4 + HIS1 ) and cwt1/cwt1 mutant on YP-K (A) or YPD-K (B) medium. 1 × 10 5 cells of each strain were spotted on YPD-K and YP-K media and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (C) Relative expression levels of CWT1 in YPD-K and YP-K media. Cells of C . albicans were spotted on YPD-K and YP-K media and cultured at 25°C for five days. (D) Relative expression levels of CWT1 in the control (GH1013cartTA) and tetON-HSF1/hsf1 on YP-K medium. Error bars, standard errors of technical duplicates. * p

    Article Snippet: Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany).

    Techniques: Mutagenesis, Cell Culture, Expressing

    Global gene-expression–profile analysis of C . albicans in the presence and absence of glucose. C . albicans cells were spotted and grown on YPD-K or YP-K media at 25°C for 60 hours. Total RNA was extracted and used for RNA-Seq assays. To be considered differentially expressed, a gene must satisfy three criteria: (1) an FPKM value higher than or equal to 20 at least in one sample, (2) a fold change value higher than or equal to 1.5, and (3) an adjusted p -value (FDR) lower than 0.05. (A) Venn diagram depicting relationships between differentially expressed genes on YPD-K (412) and YP-K (408) media and HSP90 genetic interactors (indicated in the ellipse). (B) Selected heat-shock-protein–encoding, oxidative-stress–induced, and mating-related genes up-regulated in YP-K medium. AIF1 , Apoptosis-Inducing Factor 1; BAR1 , BARrier 1; CEK1 , Candida ERK-family protein kinase; ERK, ERK-family protein kinase; FDR, false discovery rate; FPKM, fragments per kb per million reads; GPI, glycosylphosphatidylinisotol; GST3 , Glutathione S-transferase; HMX1 , HeMe oXygenase; Hsp90, Heat shock protein 90; KAR2 , KARyogamy; MAP, mitogen-activated protein; MFA1 , Mating type A1; NADH, Nicotinamide adenine dinucleotide; orf19 . 3475 , Candida gene orf19 . 3475 ; PST2 , Protoplasts-SecreTed 1; RNA-Seq, RNA sequencing; SIS1 , Slt4 Suppressor; SOD5 , SuperOxide Dismutase 5; SSA2 , Stress-Seventy subfamily A; STE4 , STErile 4; YPD-K, yeast extract-peptone-glucose-K 2 HPO 4 ; YP-K, yeast extract-peptone-K 2 HPO 4 .

    Journal: PLoS Biology

    Article Title: Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1–Hsp90 pathway

    doi: 10.1371/journal.pbio.2006966

    Figure Lengend Snippet: Global gene-expression–profile analysis of C . albicans in the presence and absence of glucose. C . albicans cells were spotted and grown on YPD-K or YP-K media at 25°C for 60 hours. Total RNA was extracted and used for RNA-Seq assays. To be considered differentially expressed, a gene must satisfy three criteria: (1) an FPKM value higher than or equal to 20 at least in one sample, (2) a fold change value higher than or equal to 1.5, and (3) an adjusted p -value (FDR) lower than 0.05. (A) Venn diagram depicting relationships between differentially expressed genes on YPD-K (412) and YP-K (408) media and HSP90 genetic interactors (indicated in the ellipse). (B) Selected heat-shock-protein–encoding, oxidative-stress–induced, and mating-related genes up-regulated in YP-K medium. AIF1 , Apoptosis-Inducing Factor 1; BAR1 , BARrier 1; CEK1 , Candida ERK-family protein kinase; ERK, ERK-family protein kinase; FDR, false discovery rate; FPKM, fragments per kb per million reads; GPI, glycosylphosphatidylinisotol; GST3 , Glutathione S-transferase; HMX1 , HeMe oXygenase; Hsp90, Heat shock protein 90; KAR2 , KARyogamy; MAP, mitogen-activated protein; MFA1 , Mating type A1; NADH, Nicotinamide adenine dinucleotide; orf19 . 3475 , Candida gene orf19 . 3475 ; PST2 , Protoplasts-SecreTed 1; RNA-Seq, RNA sequencing; SIS1 , Slt4 Suppressor; SOD5 , SuperOxide Dismutase 5; SSA2 , Stress-Seventy subfamily A; STE4 , STErile 4; YPD-K, yeast extract-peptone-glucose-K 2 HPO 4 ; YP-K, yeast extract-peptone-K 2 HPO 4 .

    Article Snippet: Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany).

    Techniques: Expressing, RNA Sequencing Assay

    MTLa2 regulates the development of mating projections in C . albicans . (A) Relative expression levels of MTL a 2 in the corresponding controls, tetON-HSF1/hsf1 , tetON-HSP90/hsp90 , and cwt1/cwt1 mutants, and MTL a 2 - overexpressing strain on YPD-K medium. Transcript levels were normalized to ACT1 . Error bars, standard errors of technical duplicates. * p

    Journal: PLoS Biology

    Article Title: Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1–Hsp90 pathway

    doi: 10.1371/journal.pbio.2006966

    Figure Lengend Snippet: MTLa2 regulates the development of mating projections in C . albicans . (A) Relative expression levels of MTL a 2 in the corresponding controls, tetON-HSF1/hsf1 , tetON-HSP90/hsp90 , and cwt1/cwt1 mutants, and MTL a 2 - overexpressing strain on YPD-K medium. Transcript levels were normalized to ACT1 . Error bars, standard errors of technical duplicates. * p

    Article Snippet: Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany).

    Techniques: Expressing