triton x 100  (Roche)


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    Structured Review

    Roche triton x 100
    Effect of N-Ethylmaleimide (NEM) on the distribution of endogenous caspase-2 (C2) (Panels A–C) and FLAG-ERα (Panel D) between nuclear and extra-nuclear (cytosol or lysate) cell fractions. (A) Caspase-2 in cytosol and nuclei of cells without (−) or with (+) NEM (20 mM) pre-treatment for 10 min prior to cell fractionation using hypotonic buffer without detergent. (B) Caspase-2 in the lysate and nuclei of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. (C) Nuclear Caspase-2 in cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to lysis using the following conditions: 1) after lysis in cell culture plates (lysis buffer added directly to the plate wells), 2) cells in suspension collected in an Eppendorf tube were instantly frozen in dry ice/ethanol, transferred to ice bath and ice-cold lysis buffer with Triton X-100 was immediately added to the frozen pellets, 3) cells in suspension collected in an Eppendorf tube at room temperature and room temperature lysis buffer with 0.5% Triton X-100 was added to the pellets. (D). FLAG-tagged ERα in the lysate and nuclei of cells without (−) and with (+) NEM (20 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. The numbers on the right of the panels reflect the gel migration of the 40 kDa, 55 kDa, and 70 kDa protein markers. To ensure that our cell lysis procedure actually reflects nuclear and extra-nuclear (LYSATE) fractions, Western blotting studies examined for Histone H1.2 as a nuclear marker (E) and Procaspase-3 as an extra-nuclear marker (F)   [31] . Cell lysis was carried out using the conditions given in (B) above.
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    Images

    1) Product Images from "A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli"

    Article Title: A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061085

    Effect of N-Ethylmaleimide (NEM) on the distribution of endogenous caspase-2 (C2) (Panels A–C) and FLAG-ERα (Panel D) between nuclear and extra-nuclear (cytosol or lysate) cell fractions. (A) Caspase-2 in cytosol and nuclei of cells without (−) or with (+) NEM (20 mM) pre-treatment for 10 min prior to cell fractionation using hypotonic buffer without detergent. (B) Caspase-2 in the lysate and nuclei of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. (C) Nuclear Caspase-2 in cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to lysis using the following conditions: 1) after lysis in cell culture plates (lysis buffer added directly to the plate wells), 2) cells in suspension collected in an Eppendorf tube were instantly frozen in dry ice/ethanol, transferred to ice bath and ice-cold lysis buffer with Triton X-100 was immediately added to the frozen pellets, 3) cells in suspension collected in an Eppendorf tube at room temperature and room temperature lysis buffer with 0.5% Triton X-100 was added to the pellets. (D). FLAG-tagged ERα in the lysate and nuclei of cells without (−) and with (+) NEM (20 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. The numbers on the right of the panels reflect the gel migration of the 40 kDa, 55 kDa, and 70 kDa protein markers. To ensure that our cell lysis procedure actually reflects nuclear and extra-nuclear (LYSATE) fractions, Western blotting studies examined for Histone H1.2 as a nuclear marker (E) and Procaspase-3 as an extra-nuclear marker (F)   [31] . Cell lysis was carried out using the conditions given in (B) above.
    Figure Legend Snippet: Effect of N-Ethylmaleimide (NEM) on the distribution of endogenous caspase-2 (C2) (Panels A–C) and FLAG-ERα (Panel D) between nuclear and extra-nuclear (cytosol or lysate) cell fractions. (A) Caspase-2 in cytosol and nuclei of cells without (−) or with (+) NEM (20 mM) pre-treatment for 10 min prior to cell fractionation using hypotonic buffer without detergent. (B) Caspase-2 in the lysate and nuclei of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. (C) Nuclear Caspase-2 in cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to lysis using the following conditions: 1) after lysis in cell culture plates (lysis buffer added directly to the plate wells), 2) cells in suspension collected in an Eppendorf tube were instantly frozen in dry ice/ethanol, transferred to ice bath and ice-cold lysis buffer with Triton X-100 was immediately added to the frozen pellets, 3) cells in suspension collected in an Eppendorf tube at room temperature and room temperature lysis buffer with 0.5% Triton X-100 was added to the pellets. (D). FLAG-tagged ERα in the lysate and nuclei of cells without (−) and with (+) NEM (20 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. The numbers on the right of the panels reflect the gel migration of the 40 kDa, 55 kDa, and 70 kDa protein markers. To ensure that our cell lysis procedure actually reflects nuclear and extra-nuclear (LYSATE) fractions, Western blotting studies examined for Histone H1.2 as a nuclear marker (E) and Procaspase-3 as an extra-nuclear marker (F) [31] . Cell lysis was carried out using the conditions given in (B) above.

    Techniques Used: Cell Fractionation, Lysis, Cell Culture, Migration, Western Blot, Marker

    2) Product Images from "PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei, Is Required for Efficient Maturation of Infection"

    Article Title: PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei, Is Required for Efficient Maturation of Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007074

    PSSA-2 requires the cytoplasmic tail for surface localisation. A. Schematic representation of PSSA-2. Predicted structural domains and signal peptide are indicated (not drawn to scale). AnTat 1.1 encodes a polypeptide of 436 amino acids, whereas the genome strain TREU 927/4 encodes a polypeptide of 425 amino acids. The sequence corresponding to the transmembrane domain is underlined; threonine residue T 305  is in boldface type and underlined. B. Immunoblot analysis of total lysates from procyclic forms of AnTat 1.1 stably transfected with plasmids encoding either an HA-tagged version of truncated PSSA-2 (ΔPSSA-2), lacking the cytoplasmic domain from residues 292–436, or full-length PSSA-2. Proteins were detected with an anti-HA antibody. 10 6  cell equivalents were loaded per lane. Markers are indicated on the left. C. Immunofluorescence analysis of HA-tagged full length PSSA-2 (top panel) and a ΔPSSA-2/GFP fusion protein (lower panel). Trypanosomes were fixed with formaldehyde and glutaraldehyde, permeabilized with Triton X-100 and stained with anti-GPEET, anti-HA or anti-BiP antibodies as indicated.
    Figure Legend Snippet: PSSA-2 requires the cytoplasmic tail for surface localisation. A. Schematic representation of PSSA-2. Predicted structural domains and signal peptide are indicated (not drawn to scale). AnTat 1.1 encodes a polypeptide of 436 amino acids, whereas the genome strain TREU 927/4 encodes a polypeptide of 425 amino acids. The sequence corresponding to the transmembrane domain is underlined; threonine residue T 305 is in boldface type and underlined. B. Immunoblot analysis of total lysates from procyclic forms of AnTat 1.1 stably transfected with plasmids encoding either an HA-tagged version of truncated PSSA-2 (ΔPSSA-2), lacking the cytoplasmic domain from residues 292–436, or full-length PSSA-2. Proteins were detected with an anti-HA antibody. 10 6 cell equivalents were loaded per lane. Markers are indicated on the left. C. Immunofluorescence analysis of HA-tagged full length PSSA-2 (top panel) and a ΔPSSA-2/GFP fusion protein (lower panel). Trypanosomes were fixed with formaldehyde and glutaraldehyde, permeabilized with Triton X-100 and stained with anti-GPEET, anti-HA or anti-BiP antibodies as indicated.

    Techniques Used: Sequencing, Stable Transfection, Transfection, Immunofluorescence, Staining

    3) Product Images from "Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C"

    Article Title: Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088154

    Increased Pgp function (as observed in the uptake assays; see   Figures 2 ,   6 , and   7 ) is associated with the transition of Pgp from detergent (Lubrol) resistant membrane (DRM) domains to detergent soluble membrane domains. Doxycycline-induced hCMEC/D3-MDR1-EGFP cells were treated with MMC (1 µM) for 2 or 4 h and analyzed at the end of the 2 h-exposure period or 20 h after the 4 h-exposure period. Cell surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin. After solubilisation of these cells with 1% (w/v) of Lubrol WX, the lysates were centrifuged at 100,000× g for 45 min at 4°C. The DRMs (pellets) resuspended in 0.5 (w/v) DOC and 0.5% (w/v) Triton X-100 and the soluble fractions (supernatant) were subjected to Neutravidin beads to isolate cell surface proteins. These were then analyzed by Western blotting using antibodies against Pgp. The protein bands were analyzed by scanning densitometry. In “A” and “B”, Pgp bands in DRMs and supernatant are presented as percentage of the control in one representative experiment. Data in “C” and “D” are shown as means ± SEM of three experiments. Asterisks denote values that significantly differed (P
    Figure Legend Snippet: Increased Pgp function (as observed in the uptake assays; see Figures 2 , 6 , and 7 ) is associated with the transition of Pgp from detergent (Lubrol) resistant membrane (DRM) domains to detergent soluble membrane domains. Doxycycline-induced hCMEC/D3-MDR1-EGFP cells were treated with MMC (1 µM) for 2 or 4 h and analyzed at the end of the 2 h-exposure period or 20 h after the 4 h-exposure period. Cell surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin. After solubilisation of these cells with 1% (w/v) of Lubrol WX, the lysates were centrifuged at 100,000× g for 45 min at 4°C. The DRMs (pellets) resuspended in 0.5 (w/v) DOC and 0.5% (w/v) Triton X-100 and the soluble fractions (supernatant) were subjected to Neutravidin beads to isolate cell surface proteins. These were then analyzed by Western blotting using antibodies against Pgp. The protein bands were analyzed by scanning densitometry. In “A” and “B”, Pgp bands in DRMs and supernatant are presented as percentage of the control in one representative experiment. Data in “C” and “D” are shown as means ± SEM of three experiments. Asterisks denote values that significantly differed (P

    Techniques Used: Western Blot

    4) Product Images from "Hyperphosphorylation and Cleavage at D421 Enhance Tau Secretion"

    Article Title: Hyperphosphorylation and Cleavage at D421 Enhance Tau Secretion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036873

    Overexpressed human tau is secreted by Hela cells. (A) No tubulin was noted in M before and after overexpression of human tau whereas tubulin staining was detected in the cell lysate (Total lysis) prepared in 6 ml of lysis buffer for comparison with the 6 ml of medium used to maintain Hela cells after transfection (arrow). In M collected from Hela cells overexpressing tau that were partially lysed (Partial Lysis) for few seconds in a solution of 0.01% Triton X-100 to induce some damage at the plasma membrane, tubulin staining became detectable (asterisk). (B) Cleaved tau was detected in M and L (lower arrow) whereas full-length tau was only detected in L (upper arrow in Total lysis). Full-length tau became detectable in M when Hela cells were partially lysed (Partial lysis) with a solution of 0.01% Triton X-100 (upper arrow). (C) Hela cells overexpressing human tau were stained with Trypan blue before being fixed to evaluate the percentage of cell death. Blue cells (arrow) corresponded to dead cells that had taken up Trypan blue.
    Figure Legend Snippet: Overexpressed human tau is secreted by Hela cells. (A) No tubulin was noted in M before and after overexpression of human tau whereas tubulin staining was detected in the cell lysate (Total lysis) prepared in 6 ml of lysis buffer for comparison with the 6 ml of medium used to maintain Hela cells after transfection (arrow). In M collected from Hela cells overexpressing tau that were partially lysed (Partial Lysis) for few seconds in a solution of 0.01% Triton X-100 to induce some damage at the plasma membrane, tubulin staining became detectable (asterisk). (B) Cleaved tau was detected in M and L (lower arrow) whereas full-length tau was only detected in L (upper arrow in Total lysis). Full-length tau became detectable in M when Hela cells were partially lysed (Partial lysis) with a solution of 0.01% Triton X-100 (upper arrow). (C) Hela cells overexpressing human tau were stained with Trypan blue before being fixed to evaluate the percentage of cell death. Blue cells (arrow) corresponded to dead cells that had taken up Trypan blue.

    Techniques Used: Over Expression, Staining, Lysis, Transfection

    5) Product Images from "A testis-specific regulator of complex and hybrid N-glycan synthesis"

    Article Title: A testis-specific regulator of complex and hybrid N-glycan synthesis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201004102

    GnT1IP-L is a type II transmembrane glycoprotein with high mannose N-glycans.  (A) GnT1IP constructs showing the N-terminal cytosolic domain of GnT1IP-L (aa 1–48; black box); the predicted transmembrane domain of GnT1IP-L (aa 49–69; white box) or signal peptide (SP) of GnT1IP-S (aa 1–26); the Golgi lumenal domain (aa 70–417 for GnT1IP-L and aa 27–373 for GnT1IP-S; gray box), and the C-terminal deletion mutants Tag-GnT1IP-S-CD1 (39 aa deletion) and -CD2 (122 aa deletion); Tag represents FLAG-HA (FL-HA), HA, or Myc; internal tags were inserted after aa 412 of GnT1IP-L (iTag 1 ) and after aa 26 of GnT1IP-S (iTag 2 ); the 48 aa stem-region deletion (Δ stem) from aa 71–118 in GnT1IP-L and aa 27–74 in GnT1IP-S is shown by a hat. The KDEL sequence was inserted after aa 373 of GnT1IP-S. (B) Lysates from CHO cells expressing HA-GnT1IP-L or FL-HA-GnT1IP-S digested with PNGase F or Endo H (+) or incubated without enzyme (−) and subjected to immunoblotting using anti-HA mAb (IB HA). (C) HeLa cells transiently expressing Myc-GnT1IP-L or GnT1IP-L-Myc-KDNYY were fixed, treated with 5 µg/ml digitonin or (D) 0.2% Triton X-100, immunolabeled for Myc-tagged GnT1IP-L (green) and actin (phalloidin; red), and observed by fluorescence microscopy. Bars, 20 µm.
    Figure Legend Snippet: GnT1IP-L is a type II transmembrane glycoprotein with high mannose N-glycans. (A) GnT1IP constructs showing the N-terminal cytosolic domain of GnT1IP-L (aa 1–48; black box); the predicted transmembrane domain of GnT1IP-L (aa 49–69; white box) or signal peptide (SP) of GnT1IP-S (aa 1–26); the Golgi lumenal domain (aa 70–417 for GnT1IP-L and aa 27–373 for GnT1IP-S; gray box), and the C-terminal deletion mutants Tag-GnT1IP-S-CD1 (39 aa deletion) and -CD2 (122 aa deletion); Tag represents FLAG-HA (FL-HA), HA, or Myc; internal tags were inserted after aa 412 of GnT1IP-L (iTag 1 ) and after aa 26 of GnT1IP-S (iTag 2 ); the 48 aa stem-region deletion (Δ stem) from aa 71–118 in GnT1IP-L and aa 27–74 in GnT1IP-S is shown by a hat. The KDEL sequence was inserted after aa 373 of GnT1IP-S. (B) Lysates from CHO cells expressing HA-GnT1IP-L or FL-HA-GnT1IP-S digested with PNGase F or Endo H (+) or incubated without enzyme (−) and subjected to immunoblotting using anti-HA mAb (IB HA). (C) HeLa cells transiently expressing Myc-GnT1IP-L or GnT1IP-L-Myc-KDNYY were fixed, treated with 5 µg/ml digitonin or (D) 0.2% Triton X-100, immunolabeled for Myc-tagged GnT1IP-L (green) and actin (phalloidin; red), and observed by fluorescence microscopy. Bars, 20 µm.

    Techniques Used: Construct, HAT Assay, Sequencing, Expressing, Incubation, Immunolabeling, Fluorescence, Microscopy

    6) Product Images from "The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein"

    Article Title: The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein

    Journal: Retrovirology

    doi: 10.1186/1742-4690-11-60

    The dUTPase activity in virions and in cell extracts.  The PPiLight pyrophosphate detection assay was performed to measure the dUTPase activity in viral extracts of WT BIV and EIAV, in Cf2Th cells that were chronically-infected by WT BIV (generating ~10 7  viral particles/ml), or in uninfected Cf2Th cells. A dUTPase activity was also tested in a complementation assay of recombinant BIV dUTPase and the extract of uninfected Cf2Th cells. The assay conditions are described in Methods and in the text. All viruses and cells were lysed and then incubated at 37°C for 30 min with dUTP-containing reaction buffer. Viral extracts were prepared from ~8.8 × 10 7  viral particles of either EIAV or WT BIV. Viruses were lysed using 0.5% Triton X-100 in the DMEM-FCS medium. Cell extracts were prepared from the pellets of ~5 × 10 4  infected or uninfected Cf2Th cells by disruption in 100 μl of 0.5% Triton X-100 in PBS for 30 minutes on ice (followed by removing the insoluble material at 15000 rpm at 4°C). The activities in cells were then tested with equal amounts of the appropriate cell extracts. The complementation assay was performed by incubating 25 ng (in 5 μl) of the purified recombinant BIV dUTPase (the 74-residues version) with 10 μl of uninfected Cf2Th cells extract (prepared from ~5 × 10 4  cells). All reactions were initiated by adding the 40 μl of dUTP-containing assay buffer for 30 min at 37°C, followed by heat inactivation, and then assayed as described in Methods. The shown values were obtained after subtracting the non-specific background produced with lysis buffer.
    Figure Legend Snippet: The dUTPase activity in virions and in cell extracts. The PPiLight pyrophosphate detection assay was performed to measure the dUTPase activity in viral extracts of WT BIV and EIAV, in Cf2Th cells that were chronically-infected by WT BIV (generating ~10 7 viral particles/ml), or in uninfected Cf2Th cells. A dUTPase activity was also tested in a complementation assay of recombinant BIV dUTPase and the extract of uninfected Cf2Th cells. The assay conditions are described in Methods and in the text. All viruses and cells were lysed and then incubated at 37°C for 30 min with dUTP-containing reaction buffer. Viral extracts were prepared from ~8.8 × 10 7 viral particles of either EIAV or WT BIV. Viruses were lysed using 0.5% Triton X-100 in the DMEM-FCS medium. Cell extracts were prepared from the pellets of ~5 × 10 4 infected or uninfected Cf2Th cells by disruption in 100 μl of 0.5% Triton X-100 in PBS for 30 minutes on ice (followed by removing the insoluble material at 15000 rpm at 4°C). The activities in cells were then tested with equal amounts of the appropriate cell extracts. The complementation assay was performed by incubating 25 ng (in 5 μl) of the purified recombinant BIV dUTPase (the 74-residues version) with 10 μl of uninfected Cf2Th cells extract (prepared from ~5 × 10 4 cells). All reactions were initiated by adding the 40 μl of dUTP-containing assay buffer for 30 min at 37°C, followed by heat inactivation, and then assayed as described in Methods. The shown values were obtained after subtracting the non-specific background produced with lysis buffer.

    Techniques Used: Activity Assay, Detection Assay, Infection, Recombinant, Incubation, Purification, Produced, Lysis

    7) Product Images from "Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates"

    Article Title: Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111277

    HDAC3 inhibitors increase both soluble and insoluble Htt-ex1s but prefer long Qs. A–F:  Indicated amounts of HDAC3 inhibitors T247, T326, and T130 were added to C3 or C4 HeLa stable cell lines. The cells were harvested after 48 h of incubation and the fraction soluble in 1% Triton X-100 was subjected to western blot analysis (A–C). The insoluble fraction was subjected to a filter trap assay (D–E). Signals were detected by anti-GFP antibodies and chemiluminescence. Signal intensities were normalized to no inhibitors (DMSO only)  = 100. The band from an anti-actin blot is shown as a loading control. Panels A, D: T247, B, E: T326, C, F: T130. *P≤0.05, **P≤0.01, ***P≤0.001 vs. 0×IC50 by ANOVA with multiple comparisons. N = 3.  G, H:  HDAC3 inhibitors do not increase Htt-ex1 mRNA levels. Effect of HDAC3 inhibitors on Htt-ex1 expression levels were assayed by qPCR. G: internal control  =  GAPDH, H: internal control  =  ACTB. Expression level was normalized to no inhibitor = 1.0. There was no statistical significance by ANOVA with multiple comparisons. N = 3.
    Figure Legend Snippet: HDAC3 inhibitors increase both soluble and insoluble Htt-ex1s but prefer long Qs. A–F: Indicated amounts of HDAC3 inhibitors T247, T326, and T130 were added to C3 or C4 HeLa stable cell lines. The cells were harvested after 48 h of incubation and the fraction soluble in 1% Triton X-100 was subjected to western blot analysis (A–C). The insoluble fraction was subjected to a filter trap assay (D–E). Signals were detected by anti-GFP antibodies and chemiluminescence. Signal intensities were normalized to no inhibitors (DMSO only)  = 100. The band from an anti-actin blot is shown as a loading control. Panels A, D: T247, B, E: T326, C, F: T130. *P≤0.05, **P≤0.01, ***P≤0.001 vs. 0×IC50 by ANOVA with multiple comparisons. N = 3. G, H: HDAC3 inhibitors do not increase Htt-ex1 mRNA levels. Effect of HDAC3 inhibitors on Htt-ex1 expression levels were assayed by qPCR. G: internal control  =  GAPDH, H: internal control  =  ACTB. Expression level was normalized to no inhibitor = 1.0. There was no statistical significance by ANOVA with multiple comparisons. N = 3.

    Techniques Used: Stable Transfection, Incubation, Western Blot, TRAP Assay, Expressing, Real-time Polymerase Chain Reaction

    8) Product Images from "GIT1 Activates p21-Activated Kinase through a Mechanism Independent of p21 Binding"

    Article Title: GIT1 Activates p21-Activated Kinase through a Mechanism Independent of p21 Binding

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.9.3849-3859.2004

    Concentration-dependent autoactivation αPAK. (A) Flag-αPAK at 0.5 or 2.5 μM as indicated was immobilized on 20 μl of anti-Flag M2 beads (corresponding to 10 or 50 μl of COS7 total cell lysates from two different experiments as indicated). The beads were incubated with 200 μM ATP in a solution containing 50 mM HEPES (pH 7.3), 10 mM MgCl 2 , 1 mM dithiothreitol, and 0.05% Triton X-100 for 15 min at 37°C, and the reaction was quenched by adding SDS-PAGE sample buffer. Samples were diluted to the same final concentration of αPAK (40 or 200 μl, respectively) for Western blot analysis with anti-phospho-S198/203 or anti-αPAK as shown. The concentration of αPAK was determined by Coomassie blue staining relative to bovine serum albumin standard. The increased immunoreactivity at higher protein concentration was quantified (as indicated) by densitometry of the bands: anti-phospho S198/203 signal at 0.5 μM αPAK is defined as 1. (B) PAK autophosphorylation does not require intact CRIB sequence. Different concentrations of Flag-αPAK or Flag-αPAK(S76P) were immobilized and allowed to undergo autophosphorylation under conditions described for A. To assess Cdc42-mediated activation, recombinant GST-Cdc42V12 (1 μM) was added with the same ATP-containing buffer and incubated as described for panel A (15 min at 37°C). Reactions were quenched by adding SDS-PAGE sample buffer. Note the appearance of lower-mobility species in parallel with anti-phosphoS198/203 signal.
    Figure Legend Snippet: Concentration-dependent autoactivation αPAK. (A) Flag-αPAK at 0.5 or 2.5 μM as indicated was immobilized on 20 μl of anti-Flag M2 beads (corresponding to 10 or 50 μl of COS7 total cell lysates from two different experiments as indicated). The beads were incubated with 200 μM ATP in a solution containing 50 mM HEPES (pH 7.3), 10 mM MgCl 2 , 1 mM dithiothreitol, and 0.05% Triton X-100 for 15 min at 37°C, and the reaction was quenched by adding SDS-PAGE sample buffer. Samples were diluted to the same final concentration of αPAK (40 or 200 μl, respectively) for Western blot analysis with anti-phospho-S198/203 or anti-αPAK as shown. The concentration of αPAK was determined by Coomassie blue staining relative to bovine serum albumin standard. The increased immunoreactivity at higher protein concentration was quantified (as indicated) by densitometry of the bands: anti-phospho S198/203 signal at 0.5 μM αPAK is defined as 1. (B) PAK autophosphorylation does not require intact CRIB sequence. Different concentrations of Flag-αPAK or Flag-αPAK(S76P) were immobilized and allowed to undergo autophosphorylation under conditions described for A. To assess Cdc42-mediated activation, recombinant GST-Cdc42V12 (1 μM) was added with the same ATP-containing buffer and incubated as described for panel A (15 min at 37°C). Reactions were quenched by adding SDS-PAGE sample buffer. Note the appearance of lower-mobility species in parallel with anti-phosphoS198/203 signal.

    Techniques Used: Concentration Assay, Incubation, SDS Page, Western Blot, Staining, Protein Concentration, Sequencing, Activation Assay, Recombinant

    Generation of β1PIX defective for GIT1 binding. (A) Schematic figure showing the location of the various protein domains in βPIX. Rat β1PIX sequences within the GIT1-binding domain are aligned to  Drosophila  dPIX sequences to identify conserved residues (in boldface type). The isoleucine 539 and glutamate 540 residues (asterisk) were selected for substitution with proline and glycine, respectively. (B) Plasmids encoding Flag-GIT1, Ha-β1PIX, and Ha-β1PIX(I539P/E540G) were expressed by in vitro translation with [ 35 S]methionine and tested for coimmunoprecipitation using anti-Flag M2 beads. Note that the wild-type protein coprecipitates very efficiently under these conditions, while the PIX mutant does not. (C) Plasmids encoding Flag-GIT1 and Ha-β1PIX or Ha-β1PIX (I539P/E540G) were coexpressed with GST-PAK in COS-7 cells and lysed with a buffer containing 1% Triton X-100. Lysates were processes as described the methods section to yield the detergent soluble (S) fraction. The pellet was washed and reextracted with 2% SDS loading buffer (P). Equal proportions of the S and P fractions were resolved by SDS-9% polyacrylamide gel electrophoresis for Western blot analysis. Both αPAK and GIT1 were predominantly located in the S fraction (lane 1 and 3); however, coexpression of GIT1 with β1PIX resulted in a shift to the pellet (lanes 5 and 6). The β1PIX(I539P/E540G) mutant when expressed with GIT1 is, by contrast, Triton X-100 soluble (lane 8).
    Figure Legend Snippet: Generation of β1PIX defective for GIT1 binding. (A) Schematic figure showing the location of the various protein domains in βPIX. Rat β1PIX sequences within the GIT1-binding domain are aligned to Drosophila dPIX sequences to identify conserved residues (in boldface type). The isoleucine 539 and glutamate 540 residues (asterisk) were selected for substitution with proline and glycine, respectively. (B) Plasmids encoding Flag-GIT1, Ha-β1PIX, and Ha-β1PIX(I539P/E540G) were expressed by in vitro translation with [ 35 S]methionine and tested for coimmunoprecipitation using anti-Flag M2 beads. Note that the wild-type protein coprecipitates very efficiently under these conditions, while the PIX mutant does not. (C) Plasmids encoding Flag-GIT1 and Ha-β1PIX or Ha-β1PIX (I539P/E540G) were coexpressed with GST-PAK in COS-7 cells and lysed with a buffer containing 1% Triton X-100. Lysates were processes as described the methods section to yield the detergent soluble (S) fraction. The pellet was washed and reextracted with 2% SDS loading buffer (P). Equal proportions of the S and P fractions were resolved by SDS-9% polyacrylamide gel electrophoresis for Western blot analysis. Both αPAK and GIT1 were predominantly located in the S fraction (lane 1 and 3); however, coexpression of GIT1 with β1PIX resulted in a shift to the pellet (lanes 5 and 6). The β1PIX(I539P/E540G) mutant when expressed with GIT1 is, by contrast, Triton X-100 soluble (lane 8).

    Techniques Used: Binding Assay, In Vitro, Mutagenesis, Polyacrylamide Gel Electrophoresis, Western Blot

    9) Product Images from "MicroRNA-31 suppresses medulloblastoma cell growth by inhibiting DNA replication through minichromosome maintenance 2"

    Article Title: MicroRNA-31 suppresses medulloblastoma cell growth by inhibiting DNA replication through minichromosome maintenance 2

    Journal: Oncotarget

    doi:

    MiR-31 regulates MCM2 function at the DNA replication origin (A) DAOY cells, transfected with MCM2 siRNA for 48 h, were harvested and the whole-cell extracts were immunoblotted with antibody against MCM2. GAPDH was shown as a loading control. (B) Flow cytometric histograms of propidium iodide-stained DAOY cells transfected with control siRNA, MCM2 siRNA for 48 h. The percentages of cells in G1, S and G2 phase of the cell cycle were determined by analysis with the Multicycle computer software. (C) Edu incorporation assays of DAOY cells transiently transfected with control siRNA or siMCM2, and (D) stable miR-31-expressing DAOY cells. The percentage of Edu positive cells was blindly calculated with counting several nonoverlaping fields. Values are means ± SD. (E) Western analysis of chromatin-bound MCM2. DAOY cells stably expressing miR-31 were fractionated into Triton-soluble (S1 fraction) and –insoluble fractions by CSK/0.5% Triton X-100 buffer. The latter fractions were extracted with DNase I and salt. The supernatant (S2 fraction, containing DNase-released chromatin-associated proteins) and pellet (P2, containing insoluble, cytoskeletal, and nuclear matrix proteins) were collected for immunoblotting assay. Bottom panel shows the relative intensity of MCM2 in S1, S2 and P2 fractions. The expression level of Triton-soluble MCM2 in MSCV cells was set to 1. (F) Fluorescence detection of chromatin-bound MDM2 in synchronized DAOY cells. Stabe DAOY cells overexpressing miR-31 were synchronized at the G0/G1 boundary by serum deprivation and thereafter were released into fresh medium containing 10% serum. At the times indicated after the release, cells were extracted with CSK/0.5% Triton X-100 buffer before fixation and then stained with α-MCM2 (red) and DAPI (blue). Right panel shows the average MCM2 immunofluorescence per nucleus of vector control and miR-31 expressing DAOY cell nuclei at the G1/S transition. Data represent averages of three independent experiments in triplicate.
    Figure Legend Snippet: MiR-31 regulates MCM2 function at the DNA replication origin (A) DAOY cells, transfected with MCM2 siRNA for 48 h, were harvested and the whole-cell extracts were immunoblotted with antibody against MCM2. GAPDH was shown as a loading control. (B) Flow cytometric histograms of propidium iodide-stained DAOY cells transfected with control siRNA, MCM2 siRNA for 48 h. The percentages of cells in G1, S and G2 phase of the cell cycle were determined by analysis with the Multicycle computer software. (C) Edu incorporation assays of DAOY cells transiently transfected with control siRNA or siMCM2, and (D) stable miR-31-expressing DAOY cells. The percentage of Edu positive cells was blindly calculated with counting several nonoverlaping fields. Values are means ± SD. (E) Western analysis of chromatin-bound MCM2. DAOY cells stably expressing miR-31 were fractionated into Triton-soluble (S1 fraction) and –insoluble fractions by CSK/0.5% Triton X-100 buffer. The latter fractions were extracted with DNase I and salt. The supernatant (S2 fraction, containing DNase-released chromatin-associated proteins) and pellet (P2, containing insoluble, cytoskeletal, and nuclear matrix proteins) were collected for immunoblotting assay. Bottom panel shows the relative intensity of MCM2 in S1, S2 and P2 fractions. The expression level of Triton-soluble MCM2 in MSCV cells was set to 1. (F) Fluorescence detection of chromatin-bound MDM2 in synchronized DAOY cells. Stabe DAOY cells overexpressing miR-31 were synchronized at the G0/G1 boundary by serum deprivation and thereafter were released into fresh medium containing 10% serum. At the times indicated after the release, cells were extracted with CSK/0.5% Triton X-100 buffer before fixation and then stained with α-MCM2 (red) and DAPI (blue). Right panel shows the average MCM2 immunofluorescence per nucleus of vector control and miR-31 expressing DAOY cell nuclei at the G1/S transition. Data represent averages of three independent experiments in triplicate.

    Techniques Used: Transfection, Flow Cytometry, Staining, Software, Expressing, Western Blot, Stable Transfection, Fluorescence, Immunofluorescence, Plasmid Preparation

    10) Product Images from "Protective Role of Phosphorylation in Turnover of Glial Fibrillary Acidic Protein in Mice"

    Article Title: Protective Role of Phosphorylation in Turnover of Glial Fibrillary Acidic Protein in Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.22-16-06972.2002

    Pulse-chase experiments of GFAP. Primary cultured astrocytes prepared from wild-type ( A , F ),  Gfap hwt/hwt ( B ,  G ), Gfap hm3/hm3  ( C , H ),  Vim −/− ( D ,  I ), and Gfap hm3/hm3 :Vim −/− ( E ,  J ) embryos were labeled for 15 min with  35 S-Met/Cys, followed by immediate harvesting or chasing for 1, 24, or 48 hr. Autoradiographs of Triton X-100-insoluble cytoskeletal fractions and immunoprecipitates from Triton X-100-soluble cytosolic fractions with anti-GFAP antibody are shown in A–E  and  F–J. K , Lower molecular weight product detected by the GFAP antibody ( asterisk ) was not detected using a head domain-specific antibody. Immunoprecipitates from Gfap hm3/hm3  astrocytes were analyzed using autoradiography ( lane 1 ), immunoblot with antibody to GFAP head domain ( lane 2 ), or immunoblot with GF 12.24 as pan-GFAP antibody ( lane 3 ).
    Figure Legend Snippet: Pulse-chase experiments of GFAP. Primary cultured astrocytes prepared from wild-type ( A , F ), Gfap hwt/hwt ( B , G ), Gfap hm3/hm3 ( C , H ), Vim −/− ( D , I ), and Gfap hm3/hm3 :Vim −/− ( E , J ) embryos were labeled for 15 min with 35 S-Met/Cys, followed by immediate harvesting or chasing for 1, 24, or 48 hr. Autoradiographs of Triton X-100-insoluble cytoskeletal fractions and immunoprecipitates from Triton X-100-soluble cytosolic fractions with anti-GFAP antibody are shown in A–E and F–J. K , Lower molecular weight product detected by the GFAP antibody ( asterisk ) was not detected using a head domain-specific antibody. Immunoprecipitates from Gfap hm3/hm3 astrocytes were analyzed using autoradiography ( lane 1 ), immunoblot with antibody to GFAP head domain ( lane 2 ), or immunoblot with GF 12.24 as pan-GFAP antibody ( lane 3 ).

    Techniques Used: Pulse Chase, Cell Culture, Labeling, Molecular Weight, Autoradiography

    11) Product Images from "Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit"

    Article Title: Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit

    Journal: Biochemical Journal

    doi: 10.1042/BJ20100757

    ERAL1 associates with the 28S mt-SSU ( A ) Left-hand panel: Western blot analysis, with the indicated antibodies, of HEK-293T cells subfractionated into nuclear (N) or cytoplasmic (Cp) compartments, with the latter fraction further divided into mitochondrial (M) and cytosolic (Cs) fractions. Equivalent volumes of each fraction are loaded. Right-hand panel: mitochondria (30 μg) were pretreated with the indicated amounts of proteinase K and solubilized with Triton X-100 or subjected directly to Western blot analysis with the indicated antibodies. ( B ) HeLa cell lysate (0.7 mg) was separated by isokinetic gradient centrifugation as detailed in the Experimental section, prior to fractionation and Western blot analysis to indicate the position of mitoribosomal subunits (DAP3 for 28S mt-SSU; MRPL3 for 39S mt-LSU). The blot is representative of three independent repeats. ( C ) Eluted immunoprecipitate from mitochondria of cells expressing MRPS27–FLAG were separated by isokinetic gradient centrifugation and fractions were silver-stained (upper panel) or subjected to Western blotting with the indicated antibodies (lower panels). 28S mt-SSU, grey circle; 39S mt-LSU, black hexagon. Gels are representative of two independent repeats.
    Figure Legend Snippet: ERAL1 associates with the 28S mt-SSU ( A ) Left-hand panel: Western blot analysis, with the indicated antibodies, of HEK-293T cells subfractionated into nuclear (N) or cytoplasmic (Cp) compartments, with the latter fraction further divided into mitochondrial (M) and cytosolic (Cs) fractions. Equivalent volumes of each fraction are loaded. Right-hand panel: mitochondria (30 μg) were pretreated with the indicated amounts of proteinase K and solubilized with Triton X-100 or subjected directly to Western blot analysis with the indicated antibodies. ( B ) HeLa cell lysate (0.7 mg) was separated by isokinetic gradient centrifugation as detailed in the Experimental section, prior to fractionation and Western blot analysis to indicate the position of mitoribosomal subunits (DAP3 for 28S mt-SSU; MRPL3 for 39S mt-LSU). The blot is representative of three independent repeats. ( C ) Eluted immunoprecipitate from mitochondria of cells expressing MRPS27–FLAG were separated by isokinetic gradient centrifugation and fractions were silver-stained (upper panel) or subjected to Western blotting with the indicated antibodies (lower panels). 28S mt-SSU, grey circle; 39S mt-LSU, black hexagon. Gels are representative of two independent repeats.

    Techniques Used: Western Blot, Gradient Centrifugation, Fractionation, Expressing, Staining

    12) Product Images from "A New Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles"

    Article Title: A New Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.104.040360

    Unilamellarity of reconstituted GUVs using a fluorescent quenching assay. Giant vesicles were prepared with a lipid mixture containing EYPC/NBD-C12-HPC (99.5:0.5 mol/mol) at a lipid/Ca 2+ -ATPase ratio of 6.5 w/w. ( a ) Unilamellarity of the GUVs was checked by measuring the distribution of fluorescent lipids between inner and outer monolayers as described in Materials and Methods. Addition of sodium hydrosulfite to reconstituted GUVs ( arrow 1 ) induced a rapid quenching of the fluorescent lipids present in the outer lipid layer. Solubilization of the vesicles by an excess of Triton X-100 ( arrow 2 ) induced a second decrease in fluorescence due to the quenching of the inner lipids. The  I 1 / I 2 . ratio is an index of the unilamellarity of the vesicles and should be equal to 0.5 for a perfect unilamellarity. The value of 0.6 found for reconstituted GUVs could be accounted for by the presence of small vesicles ( b ) or other lipidic structures ( arrows  in  c ) that were sometimes visible encapsulated by the GUVs. Scale bars of phase-contrast images correspond to 5  μ m.
    Figure Legend Snippet: Unilamellarity of reconstituted GUVs using a fluorescent quenching assay. Giant vesicles were prepared with a lipid mixture containing EYPC/NBD-C12-HPC (99.5:0.5 mol/mol) at a lipid/Ca 2+ -ATPase ratio of 6.5 w/w. ( a ) Unilamellarity of the GUVs was checked by measuring the distribution of fluorescent lipids between inner and outer monolayers as described in Materials and Methods. Addition of sodium hydrosulfite to reconstituted GUVs ( arrow 1 ) induced a rapid quenching of the fluorescent lipids present in the outer lipid layer. Solubilization of the vesicles by an excess of Triton X-100 ( arrow 2 ) induced a second decrease in fluorescence due to the quenching of the inner lipids. The I 1 / I 2 . ratio is an index of the unilamellarity of the vesicles and should be equal to 0.5 for a perfect unilamellarity. The value of 0.6 found for reconstituted GUVs could be accounted for by the presence of small vesicles ( b ) or other lipidic structures ( arrows in c ) that were sometimes visible encapsulated by the GUVs. Scale bars of phase-contrast images correspond to 5 μ m.

    Techniques Used: Fluorescence

    13) Product Images from "The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome"

    Article Title: The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20170256

    The human orthologue of bacterial RbfA associates with the mitoribosomal SSU. ( A ) Amino acid alignment (ClustalW) of RBFA from human (NP_079081.2) and  E. coli  (P0A7G2) shows identities as (*), high level of similarity by (:) and lower levels by (.). Boxed region indicates the predicted position of ‘ribosome-binding’ and RNA-binding ‘KH’ domains. The basic residues (in bold) Arg7, Arg10, Arg45, Arg80, Lys85 and Arg90 in  E. coli  RbfA are implicated in RNA binding [  21 ]. Ala75 (orange) forms an inter-helical kink shown in  B . A conserved sequence signature (I R XXLXXXXXL R XVPXLXFXXD) is located in the C-terminal region of  E. coli  RbfA. Human RBFA shares most but not all of these characteristics. ( B ) NMR-derived structures of  E. coli  RbfA (pink; PDB 1KKG, [  21 ]) and the corresponding region of human RBFA that excludes the N- and C-terminal extensions ( H. sapiens , silver; PDB 2E7G, [  53 ]) are depicted individually and superposed using Coot [  31 ]. Both exhibit type II KH domain folds (three helices and three β-strands). Human RBFA has an additional short helix, underlined in the panel  A  alignment. The inter-helical kink, formed in  E. coli  by Ala75 (1KKG) and Ser159 (2E7G) in human, is shown in orange. ( C ) Lysate (50 µg, lane 1) and mitochondria (10 µg, lanes 2–4) were prepared from HEK293 cells. Isolated mitochondria were treated with proteinase K in the absence (lane 3) or presence (lane 4) of 1% Triton X-100. Western blots detected RBFA, mitochondrial matrix markers (mitoribosomal subunits mS40 and mS25) and a cytosolic marker (cytosolic ribosomal subunit eS6). ( D ) HEK293 cell lysate (700 μg) was separated through a 10–30% sucrose gradient. Fractions were analyzed by western blot, using antibodies against mt-SSU (mS29 and mS40) and mt-LSU (uL3m and bL12m) components. The positions of the mt-SSU (28S), mt-LSU (39S) and the monosome (55S) are indicated. RBFA distribution was determined using antibodies against the endogenous protein. ( E ) FLAG-tagged mS27 was expressed, immunoprecipitated and the immunoprecipitate separated by sucrose gradient centrifugation. Fractions were subjected to western blot to detect the monosome, the mt-SSU, and its assembly intermediates (fractions 2–3). A silver-stained gel of the fractions is shown below.
    Figure Legend Snippet: The human orthologue of bacterial RbfA associates with the mitoribosomal SSU. ( A ) Amino acid alignment (ClustalW) of RBFA from human (NP_079081.2) and E. coli (P0A7G2) shows identities as (*), high level of similarity by (:) and lower levels by (.). Boxed region indicates the predicted position of ‘ribosome-binding’ and RNA-binding ‘KH’ domains. The basic residues (in bold) Arg7, Arg10, Arg45, Arg80, Lys85 and Arg90 in E. coli RbfA are implicated in RNA binding [ 21 ]. Ala75 (orange) forms an inter-helical kink shown in B . A conserved sequence signature (I R XXLXXXXXL R XVPXLXFXXD) is located in the C-terminal region of E. coli RbfA. Human RBFA shares most but not all of these characteristics. ( B ) NMR-derived structures of E. coli RbfA (pink; PDB 1KKG, [ 21 ]) and the corresponding region of human RBFA that excludes the N- and C-terminal extensions ( H. sapiens , silver; PDB 2E7G, [ 53 ]) are depicted individually and superposed using Coot [ 31 ]. Both exhibit type II KH domain folds (three helices and three β-strands). Human RBFA has an additional short helix, underlined in the panel A alignment. The inter-helical kink, formed in E. coli by Ala75 (1KKG) and Ser159 (2E7G) in human, is shown in orange. ( C ) Lysate (50 µg, lane 1) and mitochondria (10 µg, lanes 2–4) were prepared from HEK293 cells. Isolated mitochondria were treated with proteinase K in the absence (lane 3) or presence (lane 4) of 1% Triton X-100. Western blots detected RBFA, mitochondrial matrix markers (mitoribosomal subunits mS40 and mS25) and a cytosolic marker (cytosolic ribosomal subunit eS6). ( D ) HEK293 cell lysate (700 μg) was separated through a 10–30% sucrose gradient. Fractions were analyzed by western blot, using antibodies against mt-SSU (mS29 and mS40) and mt-LSU (uL3m and bL12m) components. The positions of the mt-SSU (28S), mt-LSU (39S) and the monosome (55S) are indicated. RBFA distribution was determined using antibodies against the endogenous protein. ( E ) FLAG-tagged mS27 was expressed, immunoprecipitated and the immunoprecipitate separated by sucrose gradient centrifugation. Fractions were subjected to western blot to detect the monosome, the mt-SSU, and its assembly intermediates (fractions 2–3). A silver-stained gel of the fractions is shown below.

    Techniques Used: Binding Assay, RNA Binding Assay, Sequencing, Nuclear Magnetic Resonance, Derivative Assay, Isolation, Western Blot, Marker, Immunoprecipitation, Gradient Centrifugation, Staining

    14) Product Images from "Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere"

    Article Title: Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere

    Journal:

    doi: 10.1101/gr.6022807

    RNaseOne treatment and in situ “rescue” assay. Cells cytospun on slides were permeabilized with Triton X-100 and subjected to RNaseOne treatment for 20 min (this treatment resulted in the significant delocalization of both CENPC1 and INCENP
    Figure Legend Snippet: RNaseOne treatment and in situ “rescue” assay. Cells cytospun on slides were permeabilized with Triton X-100 and subjected to RNaseOne treatment for 20 min (this treatment resulted in the significant delocalization of both CENPC1 and INCENP

    Techniques Used: In Situ

    15) Product Images from "Synaptic P-Rex1 signaling regulates hippocampal long-term depression and autism-like social behavior"

    Article Title: Synaptic P-Rex1 signaling regulates hippocampal long-term depression and autism-like social behavior

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

    doi: 10.1073/pnas.1512913112

    Interaction of PP1α with P-Rex1 and P-Rex1–mediated Rac1 activation are essential for NMDA-induced AMPAR endocytosis. ( A ) Coimmunoprecipitation of PP1α with P-Rex1 in hippocampal slices from the CA1 region treated acutely with 100 μM NMDA. ( B ) Quantification of the NMDA-induced interaction of P-Rex1 with PP1α from five independent experiments. The ratio in the WT group was defined as 1.0. ( C – I ) Hippocampal neurons expressing pCAGGS-IRES-EGFP (Veh) vector ( C  and  D ), WT P-Rex1 (P-Rex1-WT) ( E ), WT Rac1 (Rac1-WT) ( F ), P-Rex1 with the VAFA mutation (P-Rex1-VAFA) ( G ), and P-Rex1 with the GEF domain dead mutation (P-Rex1-DH dead) ( H ) were treated with 50 μM NMDA for 10 min and stained for surface HA-GluR2 (red). After Triton X-100 treatment, the neurons were stained for the total HA-GluR2 (blue). The dendritic regions marked by white squares are magnified in the bottom panels. (Scale bars, 10 μm.) ( I ) Quantification of the NMDA-induced endocytosis of surface GluR2. Data are presented as the ratio of the intensity of surface staining HA-GluR2 to the intensity of total HA-GluR2 staining. The ratio in control neurons was defined as 1.0. (WT+Veh:  n  = 12 in the control group and  n  = 12 in the NMDA group; KO+Veh:  n  = 12 in the control group and  n  = 14 in the NMDA group; KO+P-Rex1 WT:  n  = 10 in the control group and  n  = 11 in the NMDA group; KO+Rac1 WT:  n  = 11 in the control group and  n  = 12 in the NMDA group; KO+P-Rex1 VAFA:  n  = 10 in the control group and  n  = 11 in the NMDA group; KO+P-Rex1 DHdead:  n  = 12 in the control group and  n  = 13 in the NMDA group). * P
    Figure Legend Snippet: Interaction of PP1α with P-Rex1 and P-Rex1–mediated Rac1 activation are essential for NMDA-induced AMPAR endocytosis. ( A ) Coimmunoprecipitation of PP1α with P-Rex1 in hippocampal slices from the CA1 region treated acutely with 100 μM NMDA. ( B ) Quantification of the NMDA-induced interaction of P-Rex1 with PP1α from five independent experiments. The ratio in the WT group was defined as 1.0. ( C – I ) Hippocampal neurons expressing pCAGGS-IRES-EGFP (Veh) vector ( C and D ), WT P-Rex1 (P-Rex1-WT) ( E ), WT Rac1 (Rac1-WT) ( F ), P-Rex1 with the VAFA mutation (P-Rex1-VAFA) ( G ), and P-Rex1 with the GEF domain dead mutation (P-Rex1-DH dead) ( H ) were treated with 50 μM NMDA for 10 min and stained for surface HA-GluR2 (red). After Triton X-100 treatment, the neurons were stained for the total HA-GluR2 (blue). The dendritic regions marked by white squares are magnified in the bottom panels. (Scale bars, 10 μm.) ( I ) Quantification of the NMDA-induced endocytosis of surface GluR2. Data are presented as the ratio of the intensity of surface staining HA-GluR2 to the intensity of total HA-GluR2 staining. The ratio in control neurons was defined as 1.0. (WT+Veh: n = 12 in the control group and n = 12 in the NMDA group; KO+Veh: n = 12 in the control group and n = 14 in the NMDA group; KO+P-Rex1 WT: n = 10 in the control group and n = 11 in the NMDA group; KO+Rac1 WT: n = 11 in the control group and n = 12 in the NMDA group; KO+P-Rex1 VAFA: n = 10 in the control group and n = 11 in the NMDA group; KO+P-Rex1 DHdead: n = 12 in the control group and n = 13 in the NMDA group). * P

    Techniques Used: Activation Assay, Expressing, Plasmid Preparation, Mutagenesis, Staining

    16) Product Images from "BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis"

    Article Title: BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2013.09.010

    N-Terminal Change Occurs Downstream of the Release of BAK from BCL-X L (A) Schematic representation of BAK and location of Ab-1, NT, and GLY82 antibody epitopes are indicated. The trypsin cleavage site is marked by an arrow, and the resulting p19 fragment is shown. (B) The BAK N terminus became trypsin-sensitive following ABT-737 treatment. Mitochondria from ABT-737-treated Jurkat cells (5 μM for indicated times) were isolated by Dounce homogenization and subjected to limited trypsin proteolysis. Cleavage of BAK was analyzed using a panel of BAK antibodies (Gly82, NT, and Ab-1). PefablocSC (inhibitor) was included as a control. (C) The Ab-1 epitope is exposed following ABT-737 treatment. Jurkat cells were treated with ABT-737 (5 μM) or its inactive enantiomer (5 μM) for the indicated times. Cells were then labeled with BAK Ab-1/ALEXA488-labeled antibodies (open histogram) or secondary antibody alone (con, filled histogram) and analyzed for Ab-1-associated fluorescence by flow cytometry. (D) Double immunoprecipitation procedure to determine N-terminal conformation of BCL-X L -sequestered BAK. A first immunoprecipitation is directed against N-terminally changed BAK using the Ab-1 antibody. The resulting supernatant fraction is then subjected to a second immunoprecipitation against BCL-X L . (E) Ab-1 exposure occurs downstream of BAK displacement from BCL-X L . ABT-737-treated Jurkat cells (5 μM for the indicated times) were lysed in 1% CHAPS or 1% Triton X-100 and lysates subjected to double immunoprecipitation. (F) Reconstituted BAK is present as mixed population of N-terminal exposed and occluded forms. BCL-X L :BAK-reconstituted or MOCK-transfected (con) DKO MEFs were treated with ABT-737 (30 μM) for 4 hr and lysed in 1% CHAPS. Lysates were subjected to double immunoprecipitation (as detailed in D). See also  Figure S3 .
    Figure Legend Snippet: N-Terminal Change Occurs Downstream of the Release of BAK from BCL-X L (A) Schematic representation of BAK and location of Ab-1, NT, and GLY82 antibody epitopes are indicated. The trypsin cleavage site is marked by an arrow, and the resulting p19 fragment is shown. (B) The BAK N terminus became trypsin-sensitive following ABT-737 treatment. Mitochondria from ABT-737-treated Jurkat cells (5 μM for indicated times) were isolated by Dounce homogenization and subjected to limited trypsin proteolysis. Cleavage of BAK was analyzed using a panel of BAK antibodies (Gly82, NT, and Ab-1). PefablocSC (inhibitor) was included as a control. (C) The Ab-1 epitope is exposed following ABT-737 treatment. Jurkat cells were treated with ABT-737 (5 μM) or its inactive enantiomer (5 μM) for the indicated times. Cells were then labeled with BAK Ab-1/ALEXA488-labeled antibodies (open histogram) or secondary antibody alone (con, filled histogram) and analyzed for Ab-1-associated fluorescence by flow cytometry. (D) Double immunoprecipitation procedure to determine N-terminal conformation of BCL-X L -sequestered BAK. A first immunoprecipitation is directed against N-terminally changed BAK using the Ab-1 antibody. The resulting supernatant fraction is then subjected to a second immunoprecipitation against BCL-X L . (E) Ab-1 exposure occurs downstream of BAK displacement from BCL-X L . ABT-737-treated Jurkat cells (5 μM for the indicated times) were lysed in 1% CHAPS or 1% Triton X-100 and lysates subjected to double immunoprecipitation. (F) Reconstituted BAK is present as mixed population of N-terminal exposed and occluded forms. BCL-X L :BAK-reconstituted or MOCK-transfected (con) DKO MEFs were treated with ABT-737 (30 μM) for 4 hr and lysed in 1% CHAPS. Lysates were subjected to double immunoprecipitation (as detailed in D). See also Figure S3 .

    Techniques Used: Isolation, Homogenization, Labeling, Fluorescence, Flow Cytometry, Cytometry, Immunoprecipitation, Transfection

    17) Product Images from "Role of Transmembrane Domain and Cytoplasmic Tail Amino Acid Sequences of Influenza A Virus Neuraminidase in Raft Association and Virus Budding"

    Article Title: Role of Transmembrane Domain and Cytoplasmic Tail Amino Acid Sequences of Influenza A Virus Neuraminidase in Raft Association and Virus Budding

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.10.5258-5269.2004

    Triton X-100 insolubility of cell surface proteins. At 4 h p.i., influenza virus-infected (2.5 h p.i. for VSV-infected cells) MDCK cells were metabolically labeled for 60 min with 300 μCi of  35 S-protein labeling mix/ml and chased for 90 min. Cell surface proteins were biotinylated and extracted with 1% Triton X-100 on ice for 10 min. Triton X-100-soluble (S) and -insoluble (I) fractions of surface biotinylated proteins were immunoprecipitated with specific antibodies for NA, HA, or VSV G proteins, analyzed by SDS-PAGE, autoradiographed, and quantified. The percentage of Triton X-100-insoluble protein was calculated from the results from three to five independent experiments, with average variation of less than 10%. *, the Triton X-100 insolubility of HA was calculated with both HA0 and HA1 bands.
    Figure Legend Snippet: Triton X-100 insolubility of cell surface proteins. At 4 h p.i., influenza virus-infected (2.5 h p.i. for VSV-infected cells) MDCK cells were metabolically labeled for 60 min with 300 μCi of 35 S-protein labeling mix/ml and chased for 90 min. Cell surface proteins were biotinylated and extracted with 1% Triton X-100 on ice for 10 min. Triton X-100-soluble (S) and -insoluble (I) fractions of surface biotinylated proteins were immunoprecipitated with specific antibodies for NA, HA, or VSV G proteins, analyzed by SDS-PAGE, autoradiographed, and quantified. The percentage of Triton X-100-insoluble protein was calculated from the results from three to five independent experiments, with average variation of less than 10%. *, the Triton X-100 insolubility of HA was calculated with both HA0 and HA1 bands.

    Techniques Used: Infection, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page

    18) Product Images from "Fibroblast Growth Factor Inducible (Fn14)-specific Antibodies Concomitantly Display Signaling Pathway-specific Agonistic and Antagonistic Activity *"

    Article Title: Fibroblast Growth Factor Inducible (Fn14)-specific Antibodies Concomitantly Display Signaling Pathway-specific Agonistic and Antagonistic Activity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.435917

    Anti-Fn14 antibodies efficiently trigger p100 processing at low concentrations without protein G oligomerization or binding to Fc receptors. A , HT29 cells were pretreated with increasing concentrations of the two anti-Fn14 antibodies and then challenged overnight with 100 ng/ml Flag-TWEAK to sensitize cells for TNF-induced apoptosis. On the next day, cells were challenged with TNF and cycloheximide, and after an additional overnight incubation, cellular viability was determined by crystal violet staining.  B , HT29 cells were stimulated with increasing amounts of anti-Fn14 antibodies overnight with and without oligomerization by protein G (1 μg/ml) as well as with the indicated concentrations of Flag-TWEAK and Fc-Flag-TWEAK. Total cell lysates were analyzed by Western blotting with respect to the indicated proteins.  C , HT29 cells were treated for 6 h in triplicates with 1 μg/ml of PDL192 and P4A8 with and without protein G ( prot.-G ) oligomerization and with Flag-TWEAK and Fc-Flag-TWEAK (200 ng/ml). Cells were then stimulated with TNF and cycloheximide (2.5 μg/ml) to trigger TNFR1-induced cell death. After overnight incubation, cellular viability was again determined by crystal violet staining. The dose response data for TNF alone was indicated as a  dotted line  in all panels for better comparison.  D , HT29 cells were grown on glass coverslips and were stimulated the next day with the two anti-Fn14 antibodies (1 μg/ml) in the presence and absence of protein G (1 μg/ml)or with Flag-TWEAK (200 ng/ml) or Fc-Flag-TWEAK (200 ng/ml) overnight. Cells were then stained with an antibody recognizing p100/p52. Shown are representative images ( left panel ) and the ratio of nuclear to cytoplasmic fluorescence intensity ( right panel ). To calculate the ratio of nuclear to cytoplasmic fluorescence intensity, 30 cells of each group were measured.  E , HT29 and WiDr cells were challenged with Flag-TWEAK (200 ng/ml), Fc-Flag-TWEAK (200 ng/ml), and the anti-Fn14 antibodies (2 μg/ml) with and without oligomerization by protein G (0.5 μg/ml) overnight. Triton X-100 lysates were prepared and analyzed by Western blotting with respect to the indicated proteins.
    Figure Legend Snippet: Anti-Fn14 antibodies efficiently trigger p100 processing at low concentrations without protein G oligomerization or binding to Fc receptors. A , HT29 cells were pretreated with increasing concentrations of the two anti-Fn14 antibodies and then challenged overnight with 100 ng/ml Flag-TWEAK to sensitize cells for TNF-induced apoptosis. On the next day, cells were challenged with TNF and cycloheximide, and after an additional overnight incubation, cellular viability was determined by crystal violet staining. B , HT29 cells were stimulated with increasing amounts of anti-Fn14 antibodies overnight with and without oligomerization by protein G (1 μg/ml) as well as with the indicated concentrations of Flag-TWEAK and Fc-Flag-TWEAK. Total cell lysates were analyzed by Western blotting with respect to the indicated proteins. C , HT29 cells were treated for 6 h in triplicates with 1 μg/ml of PDL192 and P4A8 with and without protein G ( prot.-G ) oligomerization and with Flag-TWEAK and Fc-Flag-TWEAK (200 ng/ml). Cells were then stimulated with TNF and cycloheximide (2.5 μg/ml) to trigger TNFR1-induced cell death. After overnight incubation, cellular viability was again determined by crystal violet staining. The dose response data for TNF alone was indicated as a dotted line in all panels for better comparison. D , HT29 cells were grown on glass coverslips and were stimulated the next day with the two anti-Fn14 antibodies (1 μg/ml) in the presence and absence of protein G (1 μg/ml)or with Flag-TWEAK (200 ng/ml) or Fc-Flag-TWEAK (200 ng/ml) overnight. Cells were then stained with an antibody recognizing p100/p52. Shown are representative images ( left panel ) and the ratio of nuclear to cytoplasmic fluorescence intensity ( right panel ). To calculate the ratio of nuclear to cytoplasmic fluorescence intensity, 30 cells of each group were measured. E , HT29 and WiDr cells were challenged with Flag-TWEAK (200 ng/ml), Fc-Flag-TWEAK (200 ng/ml), and the anti-Fn14 antibodies (2 μg/ml) with and without oligomerization by protein G (0.5 μg/ml) overnight. Triton X-100 lysates were prepared and analyzed by Western blotting with respect to the indicated proteins.

    Techniques Used: Binding Assay, Incubation, Staining, Western Blot, Fluorescence

    19) Product Images from "Systemic overexpression of SQSTM1/p62 accelerates disease onset in a SOD1H46R-expressing ALS mouse model"

    Article Title: Systemic overexpression of SQSTM1/p62 accelerates disease onset in a SOD1H46R-expressing ALS mouse model

    Journal: Molecular Brain

    doi: 10.1186/s13041-018-0373-8

    Accumulation of phosphorylated SQSTM1 in the spinal cord of  SOD1 H46R -expressing mice.  a  Western blot analysis of Ser403(human)/Ser405(mouse)-phosphorylated SQSTM1 (p-S403/405), Ser349(human)/Ser351(mouse)-phosphorylated SQSTM1 (p-S349/351), LC3, and NQO1 in the spinal cord from mice with four different genotypes; wild-type (WT),  SQSTM1  (SQSTM1),  SOD1 H46R  (H46R), and  SQSTM1 ; SOD1 H46R  (SQSTM1;H46R) mice at 16 weeks of age (wk), 22 wk., and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1). Two fractions; 1% Triton X-100 soluble and 1% Triton X-100 insoluble/5%SDS soluble fractions were analyzed. GAPDH and β-actin (Actin) were used for a loading control in Triton X-100-soluble and -insoluble fractions, respectively. To properly compare the intensities of signals between phosphorylated SQSTM1 and total SQSTM1, the results from same blots for SQSTM1 in Fig.   7a , GAPDH, and Actin (asterisks) in Fig.   6a  are used again as references.  b  Quantification of 1% Triton X-100 soluble and insoluble S403/S405-phosphorylated SQSTM1.  c  Quantification of insoluble S349/S351-phosphorylated SQSTM1.  d  Quantification of the LC3-II to LC3-I ratio (LC3-II/LC3-I) in insoluble fraction.  e  Quantification of soluble NQO1.  b-e  Values are mean ± s.e.m. ( n  = 4) in an arbitrary unit relative to WT mice at 16 weeks of age. Signal intensities were normalized by the levels of GAPDH (soluble fractions) and Actin (insoluble fractions). Statistical significance was evaluated by two-way ANOVA with Bonferroni’s post hoc test (comparisons between different genotypes in the same age; * p
    Figure Legend Snippet: Accumulation of phosphorylated SQSTM1 in the spinal cord of SOD1 H46R -expressing mice. a Western blot analysis of Ser403(human)/Ser405(mouse)-phosphorylated SQSTM1 (p-S403/405), Ser349(human)/Ser351(mouse)-phosphorylated SQSTM1 (p-S349/351), LC3, and NQO1 in the spinal cord from mice with four different genotypes; wild-type (WT), SQSTM1 (SQSTM1), SOD1 H46R (H46R), and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at 16 weeks of age (wk), 22 wk., and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1). Two fractions; 1% Triton X-100 soluble and 1% Triton X-100 insoluble/5%SDS soluble fractions were analyzed. GAPDH and β-actin (Actin) were used for a loading control in Triton X-100-soluble and -insoluble fractions, respectively. To properly compare the intensities of signals between phosphorylated SQSTM1 and total SQSTM1, the results from same blots for SQSTM1 in Fig. 7a , GAPDH, and Actin (asterisks) in Fig. 6a are used again as references. b Quantification of 1% Triton X-100 soluble and insoluble S403/S405-phosphorylated SQSTM1. c Quantification of insoluble S349/S351-phosphorylated SQSTM1. d Quantification of the LC3-II to LC3-I ratio (LC3-II/LC3-I) in insoluble fraction. e Quantification of soluble NQO1. b-e Values are mean ± s.e.m. ( n  = 4) in an arbitrary unit relative to WT mice at 16 weeks of age. Signal intensities were normalized by the levels of GAPDH (soluble fractions) and Actin (insoluble fractions). Statistical significance was evaluated by two-way ANOVA with Bonferroni’s post hoc test (comparisons between different genotypes in the same age; * p

    Techniques Used: Expressing, Mouse Assay, Western Blot

    Overexpression of SQSTM1 increases misfolded SOD1 in the spinal cord of  SOD1 H46R -expressing mice.  a  Western blot analysis of SOD1.  b  Western blot analysis of misfolded SOD1.  a, b  The spinal cord samples were obtained from wild-type (WT),  SQSTM1  (SQSTM1),  SOD1 H46R  (H46R), and  SQSTM1 ; SOD1 H46R  (SQSTM1;H46R) mice at 16 weeks of age (wk), 22 wk., and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1). Two fractions; 1% Triton X-100 soluble and 1% Triton X-100 insoluble/5%SDS soluble fractions were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight forms of SOD1, respectively. SOD1(C4F6) represents misfolded SOD1. GAPDH and β-actin (Actin) were used for a loading control in Triton X-100-soluble and -insoluble fractions, respectively.  c  Quantification of 1% Triton X-100 soluble SOD1, insoluble SOD1_mono, insoluble SOD1_HMW, soluble misfolded SOD1, and insoluble misfolded SOD1. Values are mean ± s.e.m. ( n  = 4) in an arbitrary unit relative to H46R mice at 16 weeks of age. Signal intensities were normalized by the levels of GAPDH (soluble fractions) and Actin (insoluble fractions). Statistical significance was evaluated by two-way ANOVA with Bonferroni’s post hoc test (comparisons between different genotypes in the same age; * p
    Figure Legend Snippet: Overexpression of SQSTM1 increases misfolded SOD1 in the spinal cord of SOD1 H46R -expressing mice. a Western blot analysis of SOD1. b Western blot analysis of misfolded SOD1. a, b The spinal cord samples were obtained from wild-type (WT), SQSTM1 (SQSTM1), SOD1 H46R (H46R), and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at 16 weeks of age (wk), 22 wk., and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1). Two fractions; 1% Triton X-100 soluble and 1% Triton X-100 insoluble/5%SDS soluble fractions were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight forms of SOD1, respectively. SOD1(C4F6) represents misfolded SOD1. GAPDH and β-actin (Actin) were used for a loading control in Triton X-100-soluble and -insoluble fractions, respectively. c Quantification of 1% Triton X-100 soluble SOD1, insoluble SOD1_mono, insoluble SOD1_HMW, soluble misfolded SOD1, and insoluble misfolded SOD1. Values are mean ± s.e.m. ( n  = 4) in an arbitrary unit relative to H46R mice at 16 weeks of age. Signal intensities were normalized by the levels of GAPDH (soluble fractions) and Actin (insoluble fractions). Statistical significance was evaluated by two-way ANOVA with Bonferroni’s post hoc test (comparisons between different genotypes in the same age; * p

    Techniques Used: Over Expression, Expressing, Mouse Assay, Western Blot, Molecular Weight

    Overexpression of SQSTM1 increases the insoluble poly-ubqiuitinated proteins in the spinal cord of  SOD1 H46R -expressing mice.  a  Western blot analysis of SQSTM1, SQSTM1-HA (HA) and Ubiquitin (Ub) in the spinal cord from wild-type (WT),  SQSTM1  (SQSTM1),  SOD1 H46R  (H46R), and  SQSTM1 ; SOD1 H46R  (SQSTM1;H46R) mice at 16 weeks of age (wk), 22 wk., and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1). Two fractions; 1% Triton X-100 soluble and 1% Triton X-100 insoluble/5%SDS soluble fractions were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and poly-ubiquitinated proteins, respectively. GAPDH and β-actin (Actin) were used for a loading control in Triton X-100-soluble and -insoluble fractions, respectively. Since the blots used in these western analyses were same as those in Fig.   6a , same images for GAPDH and Actin (asterisks) in Fig.   6a  were used again as references.  b  Quantification of soluble and insoluble SQSTM1.  c  Quantification of soluble and insoluble poly-ubiquitinated proteins (Ub_HMW).  b, c  Values are mean ± s.e.m. ( n  = 4) in an arbitrary unit relative to WT mice at 16 weeks of age. Signal intensities were normalized by the levels of GAPDH (soluble fractions) and Actin (insoluble fractions). Statistical significance was evaluated by two-way ANOVA with Bonferroni’s post hoc test (comparisons between different genotypes in the same age; * p
    Figure Legend Snippet: Overexpression of SQSTM1 increases the insoluble poly-ubqiuitinated proteins in the spinal cord of SOD1 H46R -expressing mice. a Western blot analysis of SQSTM1, SQSTM1-HA (HA) and Ubiquitin (Ub) in the spinal cord from wild-type (WT), SQSTM1 (SQSTM1), SOD1 H46R (H46R), and SQSTM1 ; SOD1 H46R (SQSTM1;H46R) mice at 16 weeks of age (wk), 22 wk., and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1). Two fractions; 1% Triton X-100 soluble and 1% Triton X-100 insoluble/5%SDS soluble fractions were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and poly-ubiquitinated proteins, respectively. GAPDH and β-actin (Actin) were used for a loading control in Triton X-100-soluble and -insoluble fractions, respectively. Since the blots used in these western analyses were same as those in Fig. 6a , same images for GAPDH and Actin (asterisks) in Fig. 6a were used again as references. b Quantification of soluble and insoluble SQSTM1. c Quantification of soluble and insoluble poly-ubiquitinated proteins (Ub_HMW). b, c Values are mean ± s.e.m. ( n  = 4) in an arbitrary unit relative to WT mice at 16 weeks of age. Signal intensities were normalized by the levels of GAPDH (soluble fractions) and Actin (insoluble fractions). Statistical significance was evaluated by two-way ANOVA with Bonferroni’s post hoc test (comparisons between different genotypes in the same age; * p

    Techniques Used: Over Expression, Expressing, Mouse Assay, Western Blot, Molecular Weight

    20) Product Images from "Crosstalk between PKA and PKG controls pH‐dependent host cell egress of Toxoplasma gondii"

    Article Title: Crosstalk between PKA and PKG controls pH‐dependent host cell egress of Toxoplasma gondii

    Journal: The EMBO Journal

    doi: 10.15252/embj.201796794

    Cellular localisation of cAMP and cGMP signalling components possibly involved in egress A–D Localisation of PDE1‐3Ty (A), PDE2‐3Ty (B), GCα‐3Ty (C) and PKG‐3Ty (D) shown by IFA using an anti‐Ty antibody. Antibodies against GAP45, IMC1, cb‐GFP and myc‐GAP70 were used as markers of the plasma membrane, IMC, residual body and apical pole, respectively. E Western blot analysis indicates that GCα‐3Ty is present in three distinct isoforms. A PKG‐3Ty Western blot also shows three immune reactive isoforms. Catalase or actin was used as loading controls and ΔKu80 as the parental strain. F PKG‐3Ty parasites were solubilised in either PBS or 1% Triton X‐100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown. G, H The signal of Myc‐ACβ‐iKD followed by IFA (G) or Western blot (H) after 48 h ± ATc. I Invasion of ACα‐KO tachyzoites or Myc‐Acβ‐iKD after 48 h ± ATc is not affected. However, addition of ATc for 48 h reduces the invasion efficiency of ACα‐KO/Myc‐Acβ‐iKD (data are from three independent biological replicates). Error bars represent ±SD for 100 vacuoles counted in triplicate from three biological replicates. J As for destabilisation of PKAc1‐iKD or stabilisation of DDmyc‐PKArG321E‐Ty, ACα‐KO/Myc‐Acβ‐iKD tachyzoites treated with ATc for 33 or 40 h, invade and exit the monolayer of HFF cells leading to lysis, while the non‐treated parasites invade and initiate a new lytic cycle. Data information: Scale bars = 2 μm. Source data are available online for this figure.
    Figure Legend Snippet: Cellular localisation of cAMP and cGMP signalling components possibly involved in egress A–D Localisation of PDE1‐3Ty (A), PDE2‐3Ty (B), GCα‐3Ty (C) and PKG‐3Ty (D) shown by IFA using an anti‐Ty antibody. Antibodies against GAP45, IMC1, cb‐GFP and myc‐GAP70 were used as markers of the plasma membrane, IMC, residual body and apical pole, respectively. E Western blot analysis indicates that GCα‐3Ty is present in three distinct isoforms. A PKG‐3Ty Western blot also shows three immune reactive isoforms. Catalase or actin was used as loading controls and ΔKu80 as the parental strain. F PKG‐3Ty parasites were solubilised in either PBS or 1% Triton X‐100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown. G, H The signal of Myc‐ACβ‐iKD followed by IFA (G) or Western blot (H) after 48 h ± ATc. I Invasion of ACα‐KO tachyzoites or Myc‐Acβ‐iKD after 48 h ± ATc is not affected. However, addition of ATc for 48 h reduces the invasion efficiency of ACα‐KO/Myc‐Acβ‐iKD (data are from three independent biological replicates). Error bars represent ±SD for 100 vacuoles counted in triplicate from three biological replicates. J As for destabilisation of PKAc1‐iKD or stabilisation of DDmyc‐PKArG321E‐Ty, ACα‐KO/Myc‐Acβ‐iKD tachyzoites treated with ATc for 33 or 40 h, invade and exit the monolayer of HFF cells leading to lysis, while the non‐treated parasites invade and initiate a new lytic cycle. Data information: Scale bars = 2 μm. Source data are available online for this figure.

    Techniques Used: Immunofluorescence, Western Blot, Lysis

    21) Product Images from "NLRP3 inflammasome inhibitor ameliorates amyloid pathology in a mouse model of Alzheimer’s disease"

    Article Title: NLRP3 inflammasome inhibitor ameliorates amyloid pathology in a mouse model of Alzheimer’s disease

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-017-0467-9

    Effect of JC-124 on Aβ 1–42  and APP processing in TgCRND8 mice ( A–C ) ELISA of Aβ 1–42  in the TBS, TBS Triton X-100 (TBS-TX) and Guandine-HCl (GuHCl) fractions of cortical tissues from vehicle- and JC-124-treated TgCRND8 mice. Representative western blot of cortical homogenates ( D ) and quantification analysis ( E ) revealed that no changes in the full length APP as detected by 22C11 antibody but decreased levels of β-CTF as detected by 82E1 ( P =0.05, normalized to full length APP) in JC-124 treated CRND8 mice compared to vehicle-treated CRND8 mice. GAPDH was used as an internal loading control. Data represent mean ± SEM, Student’s  t -test, * P
    Figure Legend Snippet: Effect of JC-124 on Aβ 1–42 and APP processing in TgCRND8 mice ( A–C ) ELISA of Aβ 1–42 in the TBS, TBS Triton X-100 (TBS-TX) and Guandine-HCl (GuHCl) fractions of cortical tissues from vehicle- and JC-124-treated TgCRND8 mice. Representative western blot of cortical homogenates ( D ) and quantification analysis ( E ) revealed that no changes in the full length APP as detected by 22C11 antibody but decreased levels of β-CTF as detected by 82E1 ( P =0.05, normalized to full length APP) in JC-124 treated CRND8 mice compared to vehicle-treated CRND8 mice. GAPDH was used as an internal loading control. Data represent mean ± SEM, Student’s t -test, * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot

    22) Product Images from "Role of Transmembrane Domain and Cytoplasmic Tail Amino Acid Sequences of Influenza A Virus Neuraminidase in Raft Association and Virus Budding"

    Article Title: Role of Transmembrane Domain and Cytoplasmic Tail Amino Acid Sequences of Influenza A Virus Neuraminidase in Raft Association and Virus Budding

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.10.5258-5269.2004

    Triton X-100 insolubility of cell surface proteins. At 4 h p.i., influenza virus-infected (2.5 h p.i. for VSV-infected cells) MDCK cells were metabolically labeled for 60 min with 300 μCi of  35 S-protein labeling mix/ml and chased for 90 min. Cell surface proteins were biotinylated and extracted with 1% Triton X-100 on ice for 10 min. Triton X-100-soluble (S) and -insoluble (I) fractions of surface biotinylated proteins were immunoprecipitated with specific antibodies for NA, HA, or VSV G proteins, analyzed by SDS-PAGE, autoradiographed, and quantified. The percentage of Triton X-100-insoluble protein was calculated from the results from three to five independent experiments, with average variation of less than 10%. *, the Triton X-100 insolubility of HA was calculated with both HA0 and HA1 bands.
    Figure Legend Snippet: Triton X-100 insolubility of cell surface proteins. At 4 h p.i., influenza virus-infected (2.5 h p.i. for VSV-infected cells) MDCK cells were metabolically labeled for 60 min with 300 μCi of 35 S-protein labeling mix/ml and chased for 90 min. Cell surface proteins were biotinylated and extracted with 1% Triton X-100 on ice for 10 min. Triton X-100-soluble (S) and -insoluble (I) fractions of surface biotinylated proteins were immunoprecipitated with specific antibodies for NA, HA, or VSV G proteins, analyzed by SDS-PAGE, autoradiographed, and quantified. The percentage of Triton X-100-insoluble protein was calculated from the results from three to five independent experiments, with average variation of less than 10%. *, the Triton X-100 insolubility of HA was calculated with both HA0 and HA1 bands.

    Techniques Used: Infection, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page

    23) Product Images from "Apolipoprotein E Likely Contributes to a Maturation Step of Infectious Hepatitis C Virus Particles and Interacts with Viral Envelope Glycoproteins"

    Article Title: Apolipoprotein E Likely Contributes to a Maturation Step of Infectious Hepatitis C Virus Particles and Interacts with Viral Envelope Glycoproteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.01660-14

    Role of ApoE in HCV particle formation. (A) sh-ApoE and sh-NT cells were transfected with Jc1 or the assembly-deficient mutant Jc1_p7KRQQ. After 48 h, cell lysates were prepared by repetitive cycles of freezing and thawing, and envelopment of particles was probed by protease protection assay. Aliquots of each cell lysate were either mock treated, treated with 15 μg/ml proteinase K (PK) for 30 min on ice, or treated in the same way in the presence of 1% Triton X-100. The amount of PK-resistant core protein was determined by Western blotting (WB). (Upper) Representative core-specific blots are shown. (Lower) Core-specific bands detected in three independent experiments were quantified by densitometry, and values obtained with PK-treated samples were normalized to those of untreated samples. *,  P  ≤ 0.05. (B) Sedimentation profiles of core protein complexes produced in cells transfected with Jc1 or Jc1_p7KRQQ. Cell lysates were subjected to rate zonal centrifugation for 1 h at 270,000 ×  g . Twelve fractions were harvested from the top of the gradient and used for determination of density as well as core protein amount by using core ELISA. Mean values from two independent experiments are plotted. (C) Fractions of lysates from cells transfected with Jc1 (solid lines) or mock transfected (dashed lines) were used to determine amounts of ApoE and ApoCI by ELISA. Mean values from two independent experiments are plotted.
    Figure Legend Snippet: Role of ApoE in HCV particle formation. (A) sh-ApoE and sh-NT cells were transfected with Jc1 or the assembly-deficient mutant Jc1_p7KRQQ. After 48 h, cell lysates were prepared by repetitive cycles of freezing and thawing, and envelopment of particles was probed by protease protection assay. Aliquots of each cell lysate were either mock treated, treated with 15 μg/ml proteinase K (PK) for 30 min on ice, or treated in the same way in the presence of 1% Triton X-100. The amount of PK-resistant core protein was determined by Western blotting (WB). (Upper) Representative core-specific blots are shown. (Lower) Core-specific bands detected in three independent experiments were quantified by densitometry, and values obtained with PK-treated samples were normalized to those of untreated samples. *, P ≤ 0.05. (B) Sedimentation profiles of core protein complexes produced in cells transfected with Jc1 or Jc1_p7KRQQ. Cell lysates were subjected to rate zonal centrifugation for 1 h at 270,000 × g . Twelve fractions were harvested from the top of the gradient and used for determination of density as well as core protein amount by using core ELISA. Mean values from two independent experiments are plotted. (C) Fractions of lysates from cells transfected with Jc1 (solid lines) or mock transfected (dashed lines) were used to determine amounts of ApoE and ApoCI by ELISA. Mean values from two independent experiments are plotted.

    Techniques Used: Transfection, Mutagenesis, Western Blot, Sedimentation, Produced, Centrifugation, Enzyme-linked Immunosorbent Assay

    Subcellular distribution and colocalization of ApoE with viral proteins. (A) Huh7/LunetCD81H cells were infected with 30 TCID 50 /cell of Jc1. Colocalization of ApoE with viral proteins specified in the top of each panel was determined by immunofluorescence using monospecific antibodies. (B) The degree of colocalization of ApoE with given viral proteins was quantified. Each dot in the graph corresponds to one cell. Core_ring, core protein with ring-like staining pattern corresponding to lipid droplet localization; core-retic, core protein with ER-like staining pattern. (C) Huh7/LunetCD81H cells were electroporated with Jc1 RNA and fixed 48 h later. Immunofluorescence was performed using antibodies against E2, ApoE, and cellular organelle markers (PDI or the  cis -Golgi marker GM130). (D) Huh7/LunetCD81H cells were transfected with Jc1 RNA and fixed 48 h later. Cells were permeabilized with digitonin or Triton X-100, and immunofluorescence was performed using antibodies specified at the top. Insets in the lower left of merged images show magnifications of boxed areas. White arrows indicate the selected lines in the RGB line profiles shown at the right of each row. Scale bars represent 10 μm or 5 μm in regular and magnified images, respectively.
    Figure Legend Snippet: Subcellular distribution and colocalization of ApoE with viral proteins. (A) Huh7/LunetCD81H cells were infected with 30 TCID 50 /cell of Jc1. Colocalization of ApoE with viral proteins specified in the top of each panel was determined by immunofluorescence using monospecific antibodies. (B) The degree of colocalization of ApoE with given viral proteins was quantified. Each dot in the graph corresponds to one cell. Core_ring, core protein with ring-like staining pattern corresponding to lipid droplet localization; core-retic, core protein with ER-like staining pattern. (C) Huh7/LunetCD81H cells were electroporated with Jc1 RNA and fixed 48 h later. Immunofluorescence was performed using antibodies against E2, ApoE, and cellular organelle markers (PDI or the cis -Golgi marker GM130). (D) Huh7/LunetCD81H cells were transfected with Jc1 RNA and fixed 48 h later. Cells were permeabilized with digitonin or Triton X-100, and immunofluorescence was performed using antibodies specified at the top. Insets in the lower left of merged images show magnifications of boxed areas. White arrows indicate the selected lines in the RGB line profiles shown at the right of each row. Scale bars represent 10 μm or 5 μm in regular and magnified images, respectively.

    Techniques Used: Infection, Immunofluorescence, Staining, Marker, Transfection

    Presence of E2 glycoprotein in detergent-resistant membrane fractions depends on the E2 transmembrane domain. (A) Huh7/LunetCD81H cells stably expressing spE1E2 or spE1E2_DelE2TMD were lysed by treatment with 1% Triton X-100 for 30 min on ice to keep cholesterol-rich microdomains intact and fractionated by discontinuous sucrose gradient centrifugation. Fractions were harvested from the top and analyzed by E2-specific immuno-dot blotting. (B) Quantification of the immuno-dot blot. The intensity of each dot was quantified by densitometry and normalized to total E2 detected in all fractions. (C) Gradient fractions of panel A and the input were analyzed by Western blotting using antibodies specified on the left of each panel. Note that twice the amount of fraction 1 compared to fraction 10 was loaded onto the gel. Numbers on the right refer to molecular mass standards in kDa. PDI, protein disulfide isomerase; Cav-1, caveolin-1, a marker for detergent-resistant membranes. (D) sh-NT cells were transfected with Jc1 RNA, and 72 h later, cells were lysed by treatment with 0.5% Triton X-100 on ice. Samples were fractionated by discontinuous sucrose gradient centrifugation and analyzed as described for panel A. Fractions were harvested from the top and analyzed by E2-specific immuno-dot blotting, core ELISA, and ApoE ELISA. The graph represents relative quantities of E2, core, and ApoE along the gradient. ***,  P  ≤ 0.001; **,  P  ≤ 0.01; *,  P  ≤ 0.05.
    Figure Legend Snippet: Presence of E2 glycoprotein in detergent-resistant membrane fractions depends on the E2 transmembrane domain. (A) Huh7/LunetCD81H cells stably expressing spE1E2 or spE1E2_DelE2TMD were lysed by treatment with 1% Triton X-100 for 30 min on ice to keep cholesterol-rich microdomains intact and fractionated by discontinuous sucrose gradient centrifugation. Fractions were harvested from the top and analyzed by E2-specific immuno-dot blotting. (B) Quantification of the immuno-dot blot. The intensity of each dot was quantified by densitometry and normalized to total E2 detected in all fractions. (C) Gradient fractions of panel A and the input were analyzed by Western blotting using antibodies specified on the left of each panel. Note that twice the amount of fraction 1 compared to fraction 10 was loaded onto the gel. Numbers on the right refer to molecular mass standards in kDa. PDI, protein disulfide isomerase; Cav-1, caveolin-1, a marker for detergent-resistant membranes. (D) sh-NT cells were transfected with Jc1 RNA, and 72 h later, cells were lysed by treatment with 0.5% Triton X-100 on ice. Samples were fractionated by discontinuous sucrose gradient centrifugation and analyzed as described for panel A. Fractions were harvested from the top and analyzed by E2-specific immuno-dot blotting, core ELISA, and ApoE ELISA. The graph represents relative quantities of E2, core, and ApoE along the gradient. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.

    Techniques Used: Stable Transfection, Expressing, Gradient Centrifugation, Dot Blot, Western Blot, Marker, Transfection, Enzyme-linked Immunosorbent Assay

    24) Product Images from "Polysome arrest restricts miRNA turnover by preventing exosomal export of miRNA in growth-retarded mammalian cells"

    Article Title: Polysome arrest restricts miRNA turnover by preventing exosomal export of miRNA in growth-retarded mammalian cells

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-11-1521

    Exosomal export of miRNA is curtailed in cells grown to high density (A) Levels of let-7a, miR-16 and miR-24 miRNAs in FACS sorted identical number of exosomes released by HeLa and MDA-MB-231 cells grown to LDC or HDC cells (mean+/– SEM,  n  = minimum 3). (B) Amount of miRNA present in cell culture supernatant of HDC and LDC cells after removal of exosomes. (C) miRNA and AGO2 present in cell culture supernatant of HeLa cells is membrane protected. Conditioned medium from LDC state HeLa cells were treated with Triton X-100 for 15 min before they were used for exosome isolation. The estimation of miRNA was done by real time quantification and C t  values are plotted. AGO2 levels were detected by Western blot analysis. (D) Levels of CD63 exosomal marker proteins, present in cell equivalent amount of exosomes isolated from HDC and LDC state cells. (E) Relative levels of exosomal AGO2, HRS and HSP90 in exosomes isolated from LDC and HDC MDA-MB-231 cells. Absence of Dicer and β-Actin was used to rule out any cellular contamination in isolated exosomes. (F, G) Effect of CHX treatment on AGO2 and miRNA export via exosomes. Western blot for AGO2 in exosomes from DMSO or CHX treated LDC HeLa cells. CD63 levels were used to negate the variance in the release of exosomes as a consequence of CHX treatment (F). miRNA levels were quantified by real-time based quantification and Ct values were plotted (G). (H) Effect of GW4869 treatment on miRNA content of HDC and LDC state. Real time PCR based estimation of miR-122 was done with RNA from miR-122 expressing HeLa cells grown to either LDC or HDC state in the presence or absence of GW4869. ns, nonsignificant, * p
    Figure Legend Snippet: Exosomal export of miRNA is curtailed in cells grown to high density (A) Levels of let-7a, miR-16 and miR-24 miRNAs in FACS sorted identical number of exosomes released by HeLa and MDA-MB-231 cells grown to LDC or HDC cells (mean+/– SEM, n = minimum 3). (B) Amount of miRNA present in cell culture supernatant of HDC and LDC cells after removal of exosomes. (C) miRNA and AGO2 present in cell culture supernatant of HeLa cells is membrane protected. Conditioned medium from LDC state HeLa cells were treated with Triton X-100 for 15 min before they were used for exosome isolation. The estimation of miRNA was done by real time quantification and C t values are plotted. AGO2 levels were detected by Western blot analysis. (D) Levels of CD63 exosomal marker proteins, present in cell equivalent amount of exosomes isolated from HDC and LDC state cells. (E) Relative levels of exosomal AGO2, HRS and HSP90 in exosomes isolated from LDC and HDC MDA-MB-231 cells. Absence of Dicer and β-Actin was used to rule out any cellular contamination in isolated exosomes. (F, G) Effect of CHX treatment on AGO2 and miRNA export via exosomes. Western blot for AGO2 in exosomes from DMSO or CHX treated LDC HeLa cells. CD63 levels were used to negate the variance in the release of exosomes as a consequence of CHX treatment (F). miRNA levels were quantified by real-time based quantification and Ct values were plotted (G). (H) Effect of GW4869 treatment on miRNA content of HDC and LDC state. Real time PCR based estimation of miR-122 was done with RNA from miR-122 expressing HeLa cells grown to either LDC or HDC state in the presence or absence of GW4869. ns, nonsignificant, * p

    Techniques Used: FACS, Multiple Displacement Amplification, Cell Culture, Isolation, Western Blot, Marker, Real-time Polymerase Chain Reaction, Expressing

    25) Product Images from "Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release"

    Article Title: Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200607021

    Synaptotagmin-12 forms a Ca 2+ -independent complex with synaptotagmin-1.  Brain proteins were extracted in 1% of Triton X-100 and immunoprecipitated with polyclonal antibody to synaptotagmin-12 (A), monoclonal antibody to synaptotagmin-1 (B), corresponding preimmune serum or irrelevant control antibody in the presence of 1 mM Ca 2+  or EGTA. Immunoprecipitates were extensively washed and analyzed by immunoblotting with various antibodies, as indicated on the right side of each image. Input lines were loaded with 1% of total protein extract used for immunoprecipitation. (A) Synaptotagmin-1, but not SNARE, proteins are coimmunoprecipitated with synaptotagmin-12. (B) Synaptotagmin-12 and syntaxin-1 are coimmunoprecipitated with synaptotagmin-1 in a Ca 2+ -independent manner. (C–F) Synaptotagmin-1 and -12 were coimmunoprecipitated form nontreated brain homogenates or homogenates incubated with PKA activator 8-Br-cAMP (1 mM), PKA inhibitor H-89 (5 μM), or PKC activator PDBu (1 μM). Immunoblots (C and D) and protein quantifications with I 125 -labeled secondary antibodies (E and F) show the relative amounts of synaptotagmin-1 and -12 in total protein extracts and immunoprecipitations under indicated conditions. Data are shown as the mean ± the SEM.
    Figure Legend Snippet: Synaptotagmin-12 forms a Ca 2+ -independent complex with synaptotagmin-1. Brain proteins were extracted in 1% of Triton X-100 and immunoprecipitated with polyclonal antibody to synaptotagmin-12 (A), monoclonal antibody to synaptotagmin-1 (B), corresponding preimmune serum or irrelevant control antibody in the presence of 1 mM Ca 2+ or EGTA. Immunoprecipitates were extensively washed and analyzed by immunoblotting with various antibodies, as indicated on the right side of each image. Input lines were loaded with 1% of total protein extract used for immunoprecipitation. (A) Synaptotagmin-1, but not SNARE, proteins are coimmunoprecipitated with synaptotagmin-12. (B) Synaptotagmin-12 and syntaxin-1 are coimmunoprecipitated with synaptotagmin-1 in a Ca 2+ -independent manner. (C–F) Synaptotagmin-1 and -12 were coimmunoprecipitated form nontreated brain homogenates or homogenates incubated with PKA activator 8-Br-cAMP (1 mM), PKA inhibitor H-89 (5 μM), or PKC activator PDBu (1 μM). Immunoblots (C and D) and protein quantifications with I 125 -labeled secondary antibodies (E and F) show the relative amounts of synaptotagmin-1 and -12 in total protein extracts and immunoprecipitations under indicated conditions. Data are shown as the mean ± the SEM.

    Techniques Used: Immunoprecipitation, Incubation, Western Blot, Labeling

    26) Product Images from "Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion"

    Article Title: Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion

    Journal: Molecular Vision

    doi:

    Determination of the topology of myocilin in the endoplasmic reticulum. The microsomal membrane was prepared from HTM cells transduced with Ad-myocilin-GFP. The membrane fractions were untreated (-) or treated with 1% Triton X-100 (+) followed by the addition of increasing concentrations of protease K (10 μg/ml in lane 1 and 2; 20 μg/ml in lane 3 and 4; 50 μg in lane 5 and 6; 100 μg lane 7 and 8). After digestion, the membrane was probed with anti-myocilin antibody (upper panel), stripped, and re-probed with anti-calnexin antibody (lower panel).
    Figure Legend Snippet: Determination of the topology of myocilin in the endoplasmic reticulum. The microsomal membrane was prepared from HTM cells transduced with Ad-myocilin-GFP. The membrane fractions were untreated (-) or treated with 1% Triton X-100 (+) followed by the addition of increasing concentrations of protease K (10 μg/ml in lane 1 and 2; 20 μg/ml in lane 3 and 4; 50 μg in lane 5 and 6; 100 μg lane 7 and 8). After digestion, the membrane was probed with anti-myocilin antibody (upper panel), stripped, and re-probed with anti-calnexin antibody (lower panel).

    Techniques Used: Transduction

    27) Product Images from "Schip1 Is a Novel Podocyte Foot Process Protein that Mediates Actin Cytoskeleton Rearrangements and Forms a Complex with Nherf2 and Ezrin"

    Article Title: Schip1 Is a Novel Podocyte Foot Process Protein that Mediates Actin Cytoskeleton Rearrangements and Forms a Complex with Nherf2 and Ezrin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122067

    Schip1 localizes to cell lamellipodia and associates with the cortical actin cytoskeleton. (A)  Both endogenous (arrowheads, upper panel) and ectopic (arrowheads, lower panel) Schip1 localize to lamellipodia in cultured human podocytes.  (B)  To test Schip1 association to actin-rich lamellipodia regions, transiently transfected human podocytes were treated with the standard procedure (fixation and Triton X-100 permeabilization, upper panel), or incubated with saponin prior to fixation and staining for MycSchip1 (lower panel). Peripheral Schip1 expression is partially retained after saponin treatment, indicating association of the protein with detergent-insoluble cytoskeletal/plasma membrane structures. The same was not observed in control cells transfected with Stx8 (syntaxin 8).  (C)  Schip1 colocalizes with cortical F-actin in the podocyte lamellipodia. Both the Z- and XY-scanning indicate considerable signal overlap between Schip1 and F-actin along the plasma membrane in cells presenting well-developed lamellipodia.  (D)  Treatment with latrunculin A results in the dissolution of actin fibers in both control and Schip1-transfected podocytes. However, Schip1 signal remains associated with disturbed F-actin positive residues. In contrast, treatment with cytochalasin D results mostly in preservation of the cortical actin in Schip1 transfected podocytes.
    Figure Legend Snippet: Schip1 localizes to cell lamellipodia and associates with the cortical actin cytoskeleton. (A) Both endogenous (arrowheads, upper panel) and ectopic (arrowheads, lower panel) Schip1 localize to lamellipodia in cultured human podocytes. (B) To test Schip1 association to actin-rich lamellipodia regions, transiently transfected human podocytes were treated with the standard procedure (fixation and Triton X-100 permeabilization, upper panel), or incubated with saponin prior to fixation and staining for MycSchip1 (lower panel). Peripheral Schip1 expression is partially retained after saponin treatment, indicating association of the protein with detergent-insoluble cytoskeletal/plasma membrane structures. The same was not observed in control cells transfected with Stx8 (syntaxin 8). (C) Schip1 colocalizes with cortical F-actin in the podocyte lamellipodia. Both the Z- and XY-scanning indicate considerable signal overlap between Schip1 and F-actin along the plasma membrane in cells presenting well-developed lamellipodia. (D) Treatment with latrunculin A results in the dissolution of actin fibers in both control and Schip1-transfected podocytes. However, Schip1 signal remains associated with disturbed F-actin positive residues. In contrast, treatment with cytochalasin D results mostly in preservation of the cortical actin in Schip1 transfected podocytes.

    Techniques Used: Cell Culture, Transfection, Incubation, Staining, Expressing, Preserving

    28) Product Images from "Isolation of Cell Nuclei Using Inert Macromolecules to Mimic the Crowded Cytoplasm"

    Article Title: Isolation of Cell Nuclei Using Inert Macromolecules to Mimic the Crowded Cytoplasm

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007560

    Compartments in nuclei isolated in 50% Ficoll, 100 µM K-Hepes by cell lysis with 0.5% Triton X-100 to permeabilise the nuclear membrane. (A) proteins characteristic of different compartments visualised by immunofluorescence as described in   Figure 3 . Daxx is a further component of PML bodies. (B) GFP-coilin or GFP-PML isoform IV fusion proteins in nuclei of HeLa or U2OS cells, respectively. In the two left panels, approximate nuclear outlines were traced from overexposed images. Images are maximum intensity projections from deconvoluted confocal series. Bar 5 µm.
    Figure Legend Snippet: Compartments in nuclei isolated in 50% Ficoll, 100 µM K-Hepes by cell lysis with 0.5% Triton X-100 to permeabilise the nuclear membrane. (A) proteins characteristic of different compartments visualised by immunofluorescence as described in Figure 3 . Daxx is a further component of PML bodies. (B) GFP-coilin or GFP-PML isoform IV fusion proteins in nuclei of HeLa or U2OS cells, respectively. In the two left panels, approximate nuclear outlines were traced from overexposed images. Images are maximum intensity projections from deconvoluted confocal series. Bar 5 µm.

    Techniques Used: Isolation, Lysis, Immunofluorescence

    29) Product Images from "The exopolyphosphatase TbrPPX1 of Trypanosoma brucei"

    Article Title: The exopolyphosphatase TbrPPX1 of Trypanosoma brucei

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-11-4

    Subcellular localization of TbrPPX1 . Panels A-C: procyclic forms. Panels D-F: bloodstream forms. Panels A and D: c-Myc-tagged TbrPPX1; panels B and E: acidocalcisomes visualized by the VH + -PPase-antibody; panels C and F: overlay, including DAPI staining. Panel G: Detergent fractionation of bloodstream forms and procyclic cells. Pellets (P) and supernatant fractions (SN) of cells solubilized either with RIPA buffer or with 0.5% Triton X-100. Western blots were developed with monoclonal anti-c-Myc antibody (= TbrPPX1), a polyclonal antiserum against BIP, and a polyclonal antiserum against a major paraflagellar rod (PFR) protein.
    Figure Legend Snippet: Subcellular localization of TbrPPX1 . Panels A-C: procyclic forms. Panels D-F: bloodstream forms. Panels A and D: c-Myc-tagged TbrPPX1; panels B and E: acidocalcisomes visualized by the VH + -PPase-antibody; panels C and F: overlay, including DAPI staining. Panel G: Detergent fractionation of bloodstream forms and procyclic cells. Pellets (P) and supernatant fractions (SN) of cells solubilized either with RIPA buffer or with 0.5% Triton X-100. Western blots were developed with monoclonal anti-c-Myc antibody (= TbrPPX1), a polyclonal antiserum against BIP, and a polyclonal antiserum against a major paraflagellar rod (PFR) protein.

    Techniques Used: Staining, Fractionation, Western Blot

    30) Product Images from "Caveolin-1 Expression and Cavin Stability Regulate Caveolae Dynamics in Adipocyte Lipid Store Fluctuation"

    Article Title: Caveolin-1 Expression and Cavin Stability Regulate Caveolae Dynamics in Adipocyte Lipid Store Fluctuation

    Journal: Diabetes

    doi: 10.2337/db13-1961

    Caveolin-1 overexpression in 3T3-L1 increases adipocyte caveolae density.  A : Cav1-RFP transgene expression in cells transduced with retroviral constructs containing empty vector (pBabe) or a caveolin-1 cDNA fused to RFP. Membranes were immunoblotted (IB) with an anti-RFP antibody or β-actin.  B : Endogenous caveolin-1 and exogenous Cav1-RFP distribution into detergent-resistant membrane fractions. Cells stably expressing an empty pBabe vector or Cav1-RFP were lysed in the presence of cold Triton X-100. Detergent-resistant (fraction 1–5) or detergent-soluble (fraction 7–12) fractions were obtained after gradient centrifugation.  C : Fluorescent imaging of Cav1-RFP cell lines by confocal microscopy. Bar scale is 20 μm.  D  and  F : Relative mRNA expression in Cav1-RFP versus control cell lines. Indicated mRNA levels were measured by RT-QPCR and normalized to 18S or 36B4 mRNA. Values are means ± SEM obtained in three independent pools of antibiotic-selected clones.  E : Relative protein expression in Cav1-RFP versus control cell lines. Indicated proteins were assessed by Western blotting and normalized to β-actin. According to cavin nomenclature, PTRF is cavin-1, SRBC is cavin-2, and SDPR is cavin-3.  G : Electron microscopy images of 3T3-L1 adipocytes transduced with an empty vector ( upper panel ) stably expressing Cav1-RFP ( middle panel ) or adipocytes of subcutaneous adipose tissue of mice ( lower panel ). Bar scale: 300 nm.  H : Quantification of caveolae density from electron microscopy images. A total of 40-μm membrane stretches were used for caveolae quantification in each group using ImageJ software. Caveolae density is expressed as the number of invaginated caveolae per micrometer membrane length, and values are means ± SEM of 6–10 image sections. Significant differences between groups by Student  t  test are indicated as follows: *** P
    Figure Legend Snippet: Caveolin-1 overexpression in 3T3-L1 increases adipocyte caveolae density. A : Cav1-RFP transgene expression in cells transduced with retroviral constructs containing empty vector (pBabe) or a caveolin-1 cDNA fused to RFP. Membranes were immunoblotted (IB) with an anti-RFP antibody or β-actin. B : Endogenous caveolin-1 and exogenous Cav1-RFP distribution into detergent-resistant membrane fractions. Cells stably expressing an empty pBabe vector or Cav1-RFP were lysed in the presence of cold Triton X-100. Detergent-resistant (fraction 1–5) or detergent-soluble (fraction 7–12) fractions were obtained after gradient centrifugation. C : Fluorescent imaging of Cav1-RFP cell lines by confocal microscopy. Bar scale is 20 μm. D and F : Relative mRNA expression in Cav1-RFP versus control cell lines. Indicated mRNA levels were measured by RT-QPCR and normalized to 18S or 36B4 mRNA. Values are means ± SEM obtained in three independent pools of antibiotic-selected clones. E : Relative protein expression in Cav1-RFP versus control cell lines. Indicated proteins were assessed by Western blotting and normalized to β-actin. According to cavin nomenclature, PTRF is cavin-1, SRBC is cavin-2, and SDPR is cavin-3. G : Electron microscopy images of 3T3-L1 adipocytes transduced with an empty vector ( upper panel ) stably expressing Cav1-RFP ( middle panel ) or adipocytes of subcutaneous adipose tissue of mice ( lower panel ). Bar scale: 300 nm. H : Quantification of caveolae density from electron microscopy images. A total of 40-μm membrane stretches were used for caveolae quantification in each group using ImageJ software. Caveolae density is expressed as the number of invaginated caveolae per micrometer membrane length, and values are means ± SEM of 6–10 image sections. Significant differences between groups by Student t test are indicated as follows: *** P

    Techniques Used: Over Expression, Expressing, Transduction, Construct, Plasmid Preparation, Stable Transfection, Gradient Centrifugation, Imaging, Confocal Microscopy, Quantitative RT-PCR, Clone Assay, Western Blot, Electron Microscopy, Mouse Assay, Software

    31) Product Images from "Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease"

    Article Title: Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017953

    Triton X-100 solubilization of striatal lysates from A53T α-Syn mutant and age-matched control animals. Striatal lysates from A53T α-Syn mutant mice and control, non-Tg mice [4 animals per group] were extracted with Triton X-100 as described in Methods. Proteins in Triton X-100-soluble and Triton X-100-insoluble fractions were measured by Western blots. Blots show representative gels while the bar graphs are composites summarized from all animals, expressed as percent of control, non-Tg mice. (A) β-actin was added as a loading control. (B) p-GSK-3β was normalized to GSK-3β from within each fraction on the blot. (C) pSer202, pSer262 and PSer396/404 were all normalized to total Tau in each fraction, and β-actin was added as a loading control. *,  P
    Figure Legend Snippet: Triton X-100 solubilization of striatal lysates from A53T α-Syn mutant and age-matched control animals. Striatal lysates from A53T α-Syn mutant mice and control, non-Tg mice [4 animals per group] were extracted with Triton X-100 as described in Methods. Proteins in Triton X-100-soluble and Triton X-100-insoluble fractions were measured by Western blots. Blots show representative gels while the bar graphs are composites summarized from all animals, expressed as percent of control, non-Tg mice. (A) β-actin was added as a loading control. (B) p-GSK-3β was normalized to GSK-3β from within each fraction on the blot. (C) pSer202, pSer262 and PSer396/404 were all normalized to total Tau in each fraction, and β-actin was added as a loading control. *, P

    Techniques Used: Mutagenesis, Mouse Assay, Western Blot

    32) Product Images from "A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing"

    Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

    Journal: eLife

    doi: 10.7554/eLife.04137

    Cytosolic AP-2 and the Necap 1 PHear domain (residues 1–133) bind to FCHO1 and FCHO2. ( A ) Samples of ∼200 μg of GST, GST-FCHO1 (316–467), GST-FCHO2 (314–740) or GST-Sgip1 (77–214) immobilized on glutathione-Sepharose were used in pull-down assays as in with rat brain cytosol as in   Figure 9B . HC; heavy chain. The anti-Necap 1 antibody has extensive non-specific reactivity with the GST-fusion proteins (yellow asterisks) but, compared with the GST control, there is little loss of the Necap 1 from the supernatants after incubation with GST-muniscin linker fusions, as is seen for AP-2 subunits. Thus Necap 1 interacts only weakly with the FCHO1 linker segment. ( B ) Sample of ∼200 μg of GST, GST-FCHO1 (316–467) or GST-EPS15 (595–740) immobilized on glutathione-Sepharose were used in pull-down assays with rat brain cytosol alone or supplemented with 5 μM or 25 μM purified AP-2 α appendage. The marked recovery of the α appendage together with the GST-EPS15 fusion is not paralleled in the GST-FCHO1 linker pellets, and competition with soluble intact AP-2 is not evident. The reduced apparent abundance of the μ2 subunit in the pellet fractions from the GST-FCHO1 linker pull-down assays is due to comigration of the μ2 subunit with the GST fusion protein. ( C ) Samples of 250 μg of GST or 50 μg or 250 μg of GST-Necap 1 PHear domain (residues 1–133) immobilized on glutathione-Sepharose were incubated with K562 cell Triton X-100 lysate. After washing, aliquots of the supernatant (S) and pellet (P) fractions were resolved by SDS-PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose in replicate. Blots were probed with affinity-purified antibodies directed against FCHO1 or FCHO2 or with a mAb (C-8) the α subunit of AP-2. FCHO1, FCHO2 and AP-2 all show dose-dependent interactions with the Necap 1 PHear domain (  Ritter et al., 2004 ,   2007 ,   2013 ), although FCHO1 clearly displays the highest apparent affinity. In general, FCHO2 shows a weaker capacity to correct the clathrin distribution phenotype in HeLa clone #64/1.E cells, which is correlated with poorer binding to AP-2 and Necap 1. DOI: http://dx.doi.org/10.7554/eLife.04137.017
    Figure Legend Snippet: Cytosolic AP-2 and the Necap 1 PHear domain (residues 1–133) bind to FCHO1 and FCHO2. ( A ) Samples of ∼200 μg of GST, GST-FCHO1 (316–467), GST-FCHO2 (314–740) or GST-Sgip1 (77–214) immobilized on glutathione-Sepharose were used in pull-down assays as in with rat brain cytosol as in Figure 9B . HC; heavy chain. The anti-Necap 1 antibody has extensive non-specific reactivity with the GST-fusion proteins (yellow asterisks) but, compared with the GST control, there is little loss of the Necap 1 from the supernatants after incubation with GST-muniscin linker fusions, as is seen for AP-2 subunits. Thus Necap 1 interacts only weakly with the FCHO1 linker segment. ( B ) Sample of ∼200 μg of GST, GST-FCHO1 (316–467) or GST-EPS15 (595–740) immobilized on glutathione-Sepharose were used in pull-down assays with rat brain cytosol alone or supplemented with 5 μM or 25 μM purified AP-2 α appendage. The marked recovery of the α appendage together with the GST-EPS15 fusion is not paralleled in the GST-FCHO1 linker pellets, and competition with soluble intact AP-2 is not evident. The reduced apparent abundance of the μ2 subunit in the pellet fractions from the GST-FCHO1 linker pull-down assays is due to comigration of the μ2 subunit with the GST fusion protein. ( C ) Samples of 250 μg of GST or 50 μg or 250 μg of GST-Necap 1 PHear domain (residues 1–133) immobilized on glutathione-Sepharose were incubated with K562 cell Triton X-100 lysate. After washing, aliquots of the supernatant (S) and pellet (P) fractions were resolved by SDS-PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose in replicate. Blots were probed with affinity-purified antibodies directed against FCHO1 or FCHO2 or with a mAb (C-8) the α subunit of AP-2. FCHO1, FCHO2 and AP-2 all show dose-dependent interactions with the Necap 1 PHear domain ( Ritter et al., 2004 , 2007 , 2013 ), although FCHO1 clearly displays the highest apparent affinity. In general, FCHO2 shows a weaker capacity to correct the clathrin distribution phenotype in HeLa clone #64/1.E cells, which is correlated with poorer binding to AP-2 and Necap 1. DOI: http://dx.doi.org/10.7554/eLife.04137.017

    Techniques Used: Incubation, Purification, SDS Page, Staining, Affinity Purification, Binding Assay

    33) Product Images from "Genes Related to Fatty Acid β-Oxidation Play a Role in the Functional Decline of the Drosophila Brain with Age"

    Article Title: Genes Related to Fatty Acid β-Oxidation Play a Role in the Functional Decline of the Drosophila Brain with Age

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161143

    CG10814 knockdown in the nervous tissue results in a rescue of age-dependent ubiquitination profile:  (A), Ubiquitin levels (indicative of protein aggregates) detected in Triton X-100 insoluble fraction of heads from wild type Canton S aging flies, at 1, 3, 5, and 7 weeks of age and respective quantification of ubiquitin-conjugated proteins normalized to α-tubulin levels (*p
    Figure Legend Snippet: CG10814 knockdown in the nervous tissue results in a rescue of age-dependent ubiquitination profile: (A), Ubiquitin levels (indicative of protein aggregates) detected in Triton X-100 insoluble fraction of heads from wild type Canton S aging flies, at 1, 3, 5, and 7 weeks of age and respective quantification of ubiquitin-conjugated proteins normalized to α-tubulin levels (*p

    Techniques Used:

    34) Product Images from "Proprotein Convertases Process Pmel17 during Secretion *"

    Article Title: Proprotein Convertases Process Pmel17 during Secretion *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.168088

    The sequence identity of the endogenous proprotein convertase-cleavage motif within Pmel17 is not essential for proper early maturation. A , schematic representation of the IR construct.  B , IR is processed by pPCs to give rise to Mα, Mβ, and HMB45-reactive fibrillogenic fragments. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS, 1% β-mercaptoethanol + protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies.  C , IR displays a relatively normal early maturation. Cells from  B  were pulse-labeled for 30 min with  35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography ( left panel ). Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown ( right panel ).  Error bars  reflect the standard deviation from the mean of two independent experiments.  D , newly synthesized IR localizes to the ER and Golgi apparatus. Cells from  B  were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). A higher magnification of the indicated area is shown as an  inset  within each image.  E , IR is expressed at the cell surface. Cells from  B  were surface labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry ( histograms  on the  left ). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram ( right panel ).
    Figure Legend Snippet: The sequence identity of the endogenous proprotein convertase-cleavage motif within Pmel17 is not essential for proper early maturation. A , schematic representation of the IR construct. B , IR is processed by pPCs to give rise to Mα, Mβ, and HMB45-reactive fibrillogenic fragments. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS, 1% β-mercaptoethanol + protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies. C , IR displays a relatively normal early maturation. Cells from B were pulse-labeled for 30 min with 35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography ( left panel ). Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown ( right panel ). Error bars reflect the standard deviation from the mean of two independent experiments. D , newly synthesized IR localizes to the ER and Golgi apparatus. Cells from B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). A higher magnification of the indicated area is shown as an inset within each image. E , IR is expressed at the cell surface. Cells from B were surface labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry ( histograms on the left ). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram ( right panel ).

    Techniques Used: Sequencing, Construct, Derivative Assay, Western Blot, Labeling, Immunoprecipitation, Autoradiography, Pulse Chase, Standard Deviation, Synthesized, Immunofluorescence, Flow Cytometry, Cytometry

    Brefeldin A, but not monensin treatment abrogates proprotein convertase-mediated processing of Pmel17. A , monensin treatment does not abrogate proprotein convertase-mediated processing of Pmel17. Mel220 transfectants stably expressing wt-Pmel17 or IR were pulse-labeled for 30 min with  35 S and subsequently chased for the indicated times. During both labeling and chase, 10 μ m  monensin was included ( sixth  to  ninth  and  14th  to  17th lanes 6–9 ) or no inhibitor was included at all ( first  to  fifth lanes  and  10th  to  13th ). 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography.  B , quantitative PhosphorImager analysis of the pulse-chase data in  A  and a second independent experiment ( supplemental Fig. S5 ) is shown. The figure displays the ratio of the P2 form  versus  the Mα fragment at the indicated time points of chase.  Error bars  reflect the mean ± S.D. of the two independent experiments.  C , quantitative PhosphorImager analysis of the pulse-chase data in  A  and a second independent experiment ( supplemental Fig. S5 ) with maximal levels for each band set to 100% is shown.  Error bars  reflect the mean ± S.D. of two independent experiments.  D , Mel220 transfectants stably expressing wt-Pmel17 were treated or not with 10 μg/ml of brefeldin A ( BFA ) overnight and subsequently analyzed by Western blot using the Pmel17-specific antibody Pep13h.
    Figure Legend Snippet: Brefeldin A, but not monensin treatment abrogates proprotein convertase-mediated processing of Pmel17. A , monensin treatment does not abrogate proprotein convertase-mediated processing of Pmel17. Mel220 transfectants stably expressing wt-Pmel17 or IR were pulse-labeled for 30 min with 35 S and subsequently chased for the indicated times. During both labeling and chase, 10 μ m monensin was included ( sixth to ninth and 14th to 17th lanes 6–9 ) or no inhibitor was included at all ( first to fifth lanes and 10th to 13th ). 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography. B , quantitative PhosphorImager analysis of the pulse-chase data in A and a second independent experiment ( supplemental Fig. S5 ) is shown. The figure displays the ratio of the P2 form versus the Mα fragment at the indicated time points of chase. Error bars reflect the mean ± S.D. of the two independent experiments. C , quantitative PhosphorImager analysis of the pulse-chase data in A and a second independent experiment ( supplemental Fig. S5 ) with maximal levels for each band set to 100% is shown. Error bars reflect the mean ± S.D. of two independent experiments. D , Mel220 transfectants stably expressing wt-Pmel17 were treated or not with 10 μg/ml of brefeldin A ( BFA ) overnight and subsequently analyzed by Western blot using the Pmel17-specific antibody Pep13h.

    Techniques Used: Stable Transfection, Expressing, Labeling, Immunoprecipitation, Autoradiography, Pulse Chase, Western Blot

    Surface Pmel17 is already in a proprotein convertase-cleaved state. A , all Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Mel220 transfectants stably expressing wt-Pmel17 were incubated on ice with Pmel17-specific antibody HMB50, then extensively washed and lysed in 2% Triton X-100 before protein A-Sepharose beads were added to specifically immunoprecipitate the surface population of Pmel17. Immunoprecipitates ( third, fourth, seventh , and  eighth lanes ) or corresponding total cell lysates ( first, second, fifth , and  sixth lanes ) were analyzed by Western blot using antibody Pep13h. A shorter ( first  to  fourth lanes ) and a longer ( fifth  to  eighth lanes ) exposure of the same membrane is shown. The  dashed line  indicates a position where irrelevant lanes have been removed from the image. The  asterisks  indicate nonspecifically precipitated proteins. Note that the ER P1 form (present in total cell lysate, but not in the surface-IP sample) serves as an internal control in the experiment.  B , newly synthesized Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Mel220 transfectants stably expressing wt-Pmel17 were pulse-labeled for 30 min with  35 S and subsequently chased for 1 h. Antibody HMB50 was added to the intact cells on ice, before extensive washing, lysis in 2% Triton X-100, and addition of protein A-Sepharose beads to specifically immunoprecipitate the surface population of Pmel17 ( second  and  third lanes ). In parallel, a 2% Triton X-100 total cell lysate ( lane 1 ) was immunoprecipitated with Pmel17-specific antibody HMB50 as described in the legend to   Fig. 1 C . Immunoprecipitates were subsequently analyzed by autoradiography ( upper panel ). For quantification, the ratio of surface-immunoprecipitated ( lane 3 )  versus  total cell-associated Pmel17 forms (P1, P2, Mα, and Mβ) ( first lane ) was determined. Note that this percentage is almost equally low for the P2 form and the ER-located P1 form, which serves as an indicator for background precipitation ( gray shaded area ). Only Mα and Mβ are precipitated above this background.  Error bars  reflect the mean ± S.D. of two independe nt experiments. The  dashed line  indicates a position where irrelevant lanes have been removed from the image.  C , all Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Cells from  A  were surface-biotinylated at room temperature according to the protocol of the manufacturer ( left panel ) or at 4 °C to avoid any residual endocytosis during this step ( right panel ). Subsequently, biotinylated surface proteins were precipitated using avidin-agarose. Protein G-agarose was used as a specificity control. These samples ( third  and  fourth lanes  in  left panel  and  third to fifth  in  right panel ) or total cell lysates ( first  and  second lanes  in  left  and  right panels ) were analyzed by Western blot using Pmel17-specific antibodies Pep13h and Pmel-N. The  horizontal dashed lines  separate the regions of the membrane that were incubated with antibody Pmel-N ( upper part ) and antibody Pep13h ( lower part ), respectively. The  vertical dotted lines  separate longer ( right part ) or shorter ( left part ) exposures of the same membrane. Note that the ER P1 form (present in total cell lysate, but only marginally in the avidin-precipitation samples) serves as an internal control in the experiment.
    Figure Legend Snippet: Surface Pmel17 is already in a proprotein convertase-cleaved state. A , all Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Mel220 transfectants stably expressing wt-Pmel17 were incubated on ice with Pmel17-specific antibody HMB50, then extensively washed and lysed in 2% Triton X-100 before protein A-Sepharose beads were added to specifically immunoprecipitate the surface population of Pmel17. Immunoprecipitates ( third, fourth, seventh , and eighth lanes ) or corresponding total cell lysates ( first, second, fifth , and sixth lanes ) were analyzed by Western blot using antibody Pep13h. A shorter ( first to fourth lanes ) and a longer ( fifth to eighth lanes ) exposure of the same membrane is shown. The dashed line indicates a position where irrelevant lanes have been removed from the image. The asterisks indicate nonspecifically precipitated proteins. Note that the ER P1 form (present in total cell lysate, but not in the surface-IP sample) serves as an internal control in the experiment. B , newly synthesized Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Mel220 transfectants stably expressing wt-Pmel17 were pulse-labeled for 30 min with 35 S and subsequently chased for 1 h. Antibody HMB50 was added to the intact cells on ice, before extensive washing, lysis in 2% Triton X-100, and addition of protein A-Sepharose beads to specifically immunoprecipitate the surface population of Pmel17 ( second and third lanes ). In parallel, a 2% Triton X-100 total cell lysate ( lane 1 ) was immunoprecipitated with Pmel17-specific antibody HMB50 as described in the legend to Fig. 1 C . Immunoprecipitates were subsequently analyzed by autoradiography ( upper panel ). For quantification, the ratio of surface-immunoprecipitated ( lane 3 ) versus total cell-associated Pmel17 forms (P1, P2, Mα, and Mβ) ( first lane ) was determined. Note that this percentage is almost equally low for the P2 form and the ER-located P1 form, which serves as an indicator for background precipitation ( gray shaded area ). Only Mα and Mβ are precipitated above this background. Error bars reflect the mean ± S.D. of two independe nt experiments. The dashed line indicates a position where irrelevant lanes have been removed from the image. C , all Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Cells from A were surface-biotinylated at room temperature according to the protocol of the manufacturer ( left panel ) or at 4 °C to avoid any residual endocytosis during this step ( right panel ). Subsequently, biotinylated surface proteins were precipitated using avidin-agarose. Protein G-agarose was used as a specificity control. These samples ( third and fourth lanes in left panel and third to fifth in right panel ) or total cell lysates ( first and second lanes in left and right panels ) were analyzed by Western blot using Pmel17-specific antibodies Pep13h and Pmel-N. The horizontal dashed lines separate the regions of the membrane that were incubated with antibody Pmel-N ( upper part ) and antibody Pep13h ( lower part ), respectively. The vertical dotted lines separate longer ( right part ) or shorter ( left part ) exposures of the same membrane. Note that the ER P1 form (present in total cell lysate, but only marginally in the avidin-precipitation samples) serves as an internal control in the experiment.

    Techniques Used: Stable Transfection, Expressing, Incubation, Western Blot, Synthesized, Labeling, Lysis, Immunoprecipitation, Autoradiography, Avidin-Biotin Assay

    A soluble Pmel17 mutant gets secreted from cells in a proprotein convertase-cleaved form. A , schematic representation of the sPmel17-myc construct.  B , only the ER-associated P1 form can be detected for sPmel17-myc inside the cells at steady-state. Membrane lysates of Mel220 transfectants stably expressing sPmel17-myc were prepared as in   Fig. 1 B  and analyzed by Western blot using Pmel17-specific antibodies, myc-specific antibodies, or tapasin-specific antibodies for control.  C , almost all intracellular sPmel17-myc is localized to the ER. Cells from  B  were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 ( Pmel-N ), mature Pmel17 ( HMB50 ), or the myc-tag ( 9E10 ).  D , soluble sPmel17-myc gets secreted into the culture medium. Mel220 transfectants stably expressing sPmel17-myc were pulse-labeled for 30 min with  35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates ( left panel ) or culture supernatants ( right panel ) were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography. The  dashed lines  indicate positions where irrelevant lanes have been removed from the image. The  pound symbol  indicates background levels of the precipitated P1 form. The  asterisks  indicate nonspecifically precipitated proteins.  E , quantitative PhosphorImager analysis of the pulse-chase data in  D  and a second independent experiment with maximal levels for each band set to 100%.  Error bars  reflect the mean ± S.D. of these two independent experiments.
    Figure Legend Snippet: A soluble Pmel17 mutant gets secreted from cells in a proprotein convertase-cleaved form. A , schematic representation of the sPmel17-myc construct. B , only the ER-associated P1 form can be detected for sPmel17-myc inside the cells at steady-state. Membrane lysates of Mel220 transfectants stably expressing sPmel17-myc were prepared as in Fig. 1 B and analyzed by Western blot using Pmel17-specific antibodies, myc-specific antibodies, or tapasin-specific antibodies for control. C , almost all intracellular sPmel17-myc is localized to the ER. Cells from B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 ( Pmel-N ), mature Pmel17 ( HMB50 ), or the myc-tag ( 9E10 ). D , soluble sPmel17-myc gets secreted into the culture medium. Mel220 transfectants stably expressing sPmel17-myc were pulse-labeled for 30 min with 35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates ( left panel ) or culture supernatants ( right panel ) were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography. The dashed lines indicate positions where irrelevant lanes have been removed from the image. The pound symbol indicates background levels of the precipitated P1 form. The asterisks indicate nonspecifically precipitated proteins. E , quantitative PhosphorImager analysis of the pulse-chase data in D and a second independent experiment with maximal levels for each band set to 100%. Error bars reflect the mean ± S.D. of these two independent experiments.

    Techniques Used: Mutagenesis, Construct, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Synthesized, Labeling, Immunoprecipitation, Autoradiography, Pulse Chase

    35) Product Images from "Revisiting G3BP1 as a RasGAP Binding Protein: Sensitization of Tumor Cells to Chemotherapy by the RasGAP 317-326 Sequence Does Not Involve G3BP1"

    Article Title: Revisiting G3BP1 as a RasGAP Binding Protein: Sensitization of Tumor Cells to Chemotherapy by the RasGAP 317-326 Sequence Does Not Involve G3BP1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029024

    Endogenous RasGAP binds to RhoGAP but does not associate with G3BP1. Non-confluent, exponentially growing CCL39 cells (panel A) or U2OS cells (panel B) were lysed in 1% Triton X-100 lysis buffer and 1 mg of total protein extracts were immunoprecipitated with an anti-RasGAP antibody. Immunoprecipitated complexes and cell lysates (50 µg) were analyzed by Western blotting using G3BP1- and p190 RhoGAP-specific antibodies. T.L.: total lysate; asterisks: non-specific bands; #: immunoglobulin heavy chains.
    Figure Legend Snippet: Endogenous RasGAP binds to RhoGAP but does not associate with G3BP1. Non-confluent, exponentially growing CCL39 cells (panel A) or U2OS cells (panel B) were lysed in 1% Triton X-100 lysis buffer and 1 mg of total protein extracts were immunoprecipitated with an anti-RasGAP antibody. Immunoprecipitated complexes and cell lysates (50 µg) were analyzed by Western blotting using G3BP1- and p190 RhoGAP-specific antibodies. T.L.: total lysate; asterisks: non-specific bands; #: immunoglobulin heavy chains.

    Techniques Used: Lysis, Immunoprecipitation, Western Blot

    36) Product Images from "Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release"

    Article Title: Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200607021

    Synaptotagmin-12 forms a Ca 2+ -independent complex with synaptotagmin-1.  Brain proteins were extracted in 1% of Triton X-100 and immunoprecipitated with polyclonal antibody to synaptotagmin-12 (A), monoclonal antibody to synaptotagmin-1 (B), corresponding preimmune serum or irrelevant control antibody in the presence of 1 mM Ca 2+  or EGTA. Immunoprecipitates were extensively washed and analyzed by immunoblotting with various antibodies, as indicated on the right side of each image. Input lines were loaded with 1% of total protein extract used for immunoprecipitation. (A) Synaptotagmin-1, but not SNARE, proteins are coimmunoprecipitated with synaptotagmin-12. (B) Synaptotagmin-12 and syntaxin-1 are coimmunoprecipitated with synaptotagmin-1 in a Ca 2+ -independent manner. (C–F) Synaptotagmin-1 and -12 were coimmunoprecipitated form nontreated brain homogenates or homogenates incubated with PKA activator 8-Br-cAMP (1 mM), PKA inhibitor H-89 (5 μM), or PKC activator PDBu (1 μM). Immunoblots (C and D) and protein quantifications with I 125 -labeled secondary antibodies (E and F) show the relative amounts of synaptotagmin-1 and -12 in total protein extracts and immunoprecipitations under indicated conditions. Data are shown as the mean ± the SEM.
    Figure Legend Snippet: Synaptotagmin-12 forms a Ca 2+ -independent complex with synaptotagmin-1. Brain proteins were extracted in 1% of Triton X-100 and immunoprecipitated with polyclonal antibody to synaptotagmin-12 (A), monoclonal antibody to synaptotagmin-1 (B), corresponding preimmune serum or irrelevant control antibody in the presence of 1 mM Ca 2+ or EGTA. Immunoprecipitates were extensively washed and analyzed by immunoblotting with various antibodies, as indicated on the right side of each image. Input lines were loaded with 1% of total protein extract used for immunoprecipitation. (A) Synaptotagmin-1, but not SNARE, proteins are coimmunoprecipitated with synaptotagmin-12. (B) Synaptotagmin-12 and syntaxin-1 are coimmunoprecipitated with synaptotagmin-1 in a Ca 2+ -independent manner. (C–F) Synaptotagmin-1 and -12 were coimmunoprecipitated form nontreated brain homogenates or homogenates incubated with PKA activator 8-Br-cAMP (1 mM), PKA inhibitor H-89 (5 μM), or PKC activator PDBu (1 μM). Immunoblots (C and D) and protein quantifications with I 125 -labeled secondary antibodies (E and F) show the relative amounts of synaptotagmin-1 and -12 in total protein extracts and immunoprecipitations under indicated conditions. Data are shown as the mean ± the SEM.

    Techniques Used: Immunoprecipitation, Incubation, Western Blot, Labeling

    37) Product Images from "NMDA Receptor Function and NMDA Receptor-Dependent Phosphorylation of Huntingtin Is Altered by the Endocytic Protein HIP1"

    Article Title: NMDA Receptor Function and NMDA Receptor-Dependent Phosphorylation of Huntingtin Is Altered by the Endocytic Protein HIP1

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5175-06.2007

    HIP1 interacts with NMDARs  in vitro  and in brain lysate.  A , Soluble proteins from brain lysate were affinity purified with either GST alone or GST-HIP1-(219–616) bound to glutathione-Sepharose beads. The input amount of GST alone and GST fusion protein was verified by Coomassie blue staining, and their sizes were compared with a molecular-weight (MW) marker. Bound proteins were eluted and analyzed by Western blot and probed with anti-NR2A and anti-NR2B Abs as indicated.  B , Brains from 3-month-old wild-type and HIP1 −/−  mice were homogenized and solubilized in the presence of 0.2% SDS and 0.8% Triton X-100. Proteins were immunoprecipitated with mAb HIP1#9 or normal IgG, separated by Western blot, and probed with Abs against HIP1 and NR2B. The same amount of sample was loaded on gels stained for NR2B and HIP1.  C , Proteins were immunoprecipitated as described in  B  with an Ab against NR2B and normal IgG.  D , Recombinant HIP1-(219–616)-His 6  and recombinant PSD-95-PDZ2-His 6  were expressed and purified from bacterial lysate. Twenty-five micrograms of purified HIP1 or PDZ2 were incubated with GST alone, GST-NR2A-(1017–1464), and GST-NR2B-(1001–1484) bound to glutathione-Sepharose beads. The input amount of GST alone and GST fusion proteins was verified by Coomassie blue staining, and their sizes were compared with a molecular-weight marker. The amount of specifically bound protein was eluted, immunoblotted, and probed with mAb HIP1#9 or an anti-His Ab, demonstrating direct interaction between HIP1 and NMDARs and PDZ2 and NMDARs, respectively. IP, Immunoprecipitate.
    Figure Legend Snippet: HIP1 interacts with NMDARs in vitro and in brain lysate. A , Soluble proteins from brain lysate were affinity purified with either GST alone or GST-HIP1-(219–616) bound to glutathione-Sepharose beads. The input amount of GST alone and GST fusion protein was verified by Coomassie blue staining, and their sizes were compared with a molecular-weight (MW) marker. Bound proteins were eluted and analyzed by Western blot and probed with anti-NR2A and anti-NR2B Abs as indicated. B , Brains from 3-month-old wild-type and HIP1 −/− mice were homogenized and solubilized in the presence of 0.2% SDS and 0.8% Triton X-100. Proteins were immunoprecipitated with mAb HIP1#9 or normal IgG, separated by Western blot, and probed with Abs against HIP1 and NR2B. The same amount of sample was loaded on gels stained for NR2B and HIP1. C , Proteins were immunoprecipitated as described in B with an Ab against NR2B and normal IgG. D , Recombinant HIP1-(219–616)-His 6 and recombinant PSD-95-PDZ2-His 6 were expressed and purified from bacterial lysate. Twenty-five micrograms of purified HIP1 or PDZ2 were incubated with GST alone, GST-NR2A-(1017–1464), and GST-NR2B-(1001–1484) bound to glutathione-Sepharose beads. The input amount of GST alone and GST fusion proteins was verified by Coomassie blue staining, and their sizes were compared with a molecular-weight marker. The amount of specifically bound protein was eluted, immunoblotted, and probed with mAb HIP1#9 or an anti-His Ab, demonstrating direct interaction between HIP1 and NMDARs and PDZ2 and NMDARs, respectively. IP, Immunoprecipitate.

    Techniques Used: In Vitro, Affinity Purification, Staining, Molecular Weight, Marker, Western Blot, Mouse Assay, Immunoprecipitation, Recombinant, Purification, Incubation

    38) Product Images from "Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1‐linked amyotrophic lateral sclerosis mice"

    Article Title: Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1‐linked amyotrophic lateral sclerosis mice

    Journal: Journal of Neurochemistry

    doi: 10.1111/jnc.14285

    Administration of Cystatin C (CysC) induces its aggregation in lumbar motor neurons. (a and b) CysC aggregation in the motor neurons of CysC‐administered SOD1 G93A  mice. CysC aggregates were found in the motor neurons of CysC‐administered mice. The motor neurons were immunostained by the antibodies for ChAT (red) and CysC (green) (a). Averaged number of CysC aggregates in ChAT‐positive motor neurons was plotted against the aggregate diameter (b). A total of 30 aggregates each in the lumbar spinal cords from three CysC‐administered SOD1 G93A  mice were measured. (c and d) CysC aggregates did not co‐localize with p62 or poly‐ubiquitin (poly‐Ubi). The lack of co‐localization was confirmed by a fluorescence intensity profile (d), where indicated by an arrow in (c). (e) Eosinophilic inclusions found in the ventral horn neurons of the CysC‐administered mice. (f–h) Immunoblotting analyses of CysC aggregation. In the CysC‐administered SOD1 G93A  mouse spinal cords, detergent (Triton X‐100)‐soluble monomeric CysC was decreased (g), whereas insoluble tetrameric CysC was accumulated (h). The amounts of CysC were normalized to β‐actin, and are shown as mean ± SEM. Scale bars: 20 μm.
    Figure Legend Snippet: Administration of Cystatin C (CysC) induces its aggregation in lumbar motor neurons. (a and b) CysC aggregation in the motor neurons of CysC‐administered SOD1 G93A mice. CysC aggregates were found in the motor neurons of CysC‐administered mice. The motor neurons were immunostained by the antibodies for ChAT (red) and CysC (green) (a). Averaged number of CysC aggregates in ChAT‐positive motor neurons was plotted against the aggregate diameter (b). A total of 30 aggregates each in the lumbar spinal cords from three CysC‐administered SOD1 G93A mice were measured. (c and d) CysC aggregates did not co‐localize with p62 or poly‐ubiquitin (poly‐Ubi). The lack of co‐localization was confirmed by a fluorescence intensity profile (d), where indicated by an arrow in (c). (e) Eosinophilic inclusions found in the ventral horn neurons of the CysC‐administered mice. (f–h) Immunoblotting analyses of CysC aggregation. In the CysC‐administered SOD1 G93A mouse spinal cords, detergent (Triton X‐100)‐soluble monomeric CysC was decreased (g), whereas insoluble tetrameric CysC was accumulated (h). The amounts of CysC were normalized to β‐actin, and are shown as mean ± SEM. Scale bars: 20 μm.

    Techniques Used: Mouse Assay, Fluorescence

    Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A  mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A  mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A  mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A  mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to LC3‐II were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A  mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).
    Figure Legend Snippet: Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to LC3‐II were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).

    Techniques Used: Mouse Assay, Staining, Derivative Assay

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    Isolation:

    Article Title: Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures
    Article Snippet: The cell death was assessed 24 h later with the trypan blue exclusion test by incubation with 0.05% (v/v) trypan blue in PBS for 5 min. Stained neurons (i.e., death neurons) were quantified in random images taken with a phase-contrast microscope (containing 150 to 200 cells). .. Isolation of a Triton-Insoluble Biochemical Fraction Homogenates of cell cultures were recovered in a buffer containing 5 mM Tris-Cl, 1% Triton X-100 and a mixture of protease inhibitors (Complete, Roche) to separate the detergent-insoluble fraction (i.e., enriched in postsynaptic densities and lipid rafts) after centrifugation at 100,000 ×g for 1 h. The pellet was resuspended in 50 mM Hepes pH 7.4. .. Isolation of a Crude Membrane Fraction (P2) from Wild Type and Knockout Mice Tissue homogenates from the cerebellum and forebrain were used to obtain a crude membrane fraction (P2) by differential centrifugation steps as reported ( ).

    Centrifugation:

    Article Title: Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures
    Article Snippet: The cell death was assessed 24 h later with the trypan blue exclusion test by incubation with 0.05% (v/v) trypan blue in PBS for 5 min. Stained neurons (i.e., death neurons) were quantified in random images taken with a phase-contrast microscope (containing 150 to 200 cells). .. Isolation of a Triton-Insoluble Biochemical Fraction Homogenates of cell cultures were recovered in a buffer containing 5 mM Tris-Cl, 1% Triton X-100 and a mixture of protease inhibitors (Complete, Roche) to separate the detergent-insoluble fraction (i.e., enriched in postsynaptic densities and lipid rafts) after centrifugation at 100,000 ×g for 1 h. The pellet was resuspended in 50 mM Hepes pH 7.4. .. Isolation of a Crude Membrane Fraction (P2) from Wild Type and Knockout Mice Tissue homogenates from the cerebellum and forebrain were used to obtain a crude membrane fraction (P2) by differential centrifugation steps as reported ( ).

    Incubation:

    Article Title: Nuclear-to-cytoplasmic Relocalization of the Proliferating Cell Nuclear Antigen (PCNA) during Differentiation Involves a Chromosome Region Maintenance 1 (CRM1)-dependent Export and Is a Prerequisite for PCNA Antiapoptotic Activity in Mature Neutrophils *
    Article Snippet: PCNA immunofluorescence analysis of HL60, PLB985, and human neutrophils was performed after cytocentrifugation as described previously ( ). .. Briefly, cells were fixed in PBS containing 3.7% formaldehyde (Sigma) for 20 min on ice and permeabilized with Triton X-100 (0.25%) for 5 min at room temperature followed by ice-cold methanol for 10 min, incubated with the rabbit polyclonal anti-PCNA (diluted 1/50, clone Ab5 Calbiochem) for 45 min followed by biotinylated rabbit IgG, diluted 1:100 (Dako Cytomation) for 30 min, and then followed by streptavidin-coupled Alexa Fluor 555, diluted 1:200 (2 μg/ml, Molecular Probes) for 30 min. For HA or CRM1 detection, HeLa cells were fixed as described for PCNA labeling and were incubated with a rabbit polyclonal anti-HA (diluted 1:100, Roche Applied Science) or anti-CRM1 (Santa Cruz Biotechnology) antibody, respectively, followed by an Alexa Fluor 555-coupled goat-anti rabbit IgG (diluted 1:100, Molecular Probes). .. The analysis of the subcellular localization of the NES1-EGFP and NES2-EGFP and EGFP-NES1 and EGFP-NES2 in HeLa cells was performed 24 h after transfection.

    Labeling:

    Article Title: Nuclear-to-cytoplasmic Relocalization of the Proliferating Cell Nuclear Antigen (PCNA) during Differentiation Involves a Chromosome Region Maintenance 1 (CRM1)-dependent Export and Is a Prerequisite for PCNA Antiapoptotic Activity in Mature Neutrophils *
    Article Snippet: PCNA immunofluorescence analysis of HL60, PLB985, and human neutrophils was performed after cytocentrifugation as described previously ( ). .. Briefly, cells were fixed in PBS containing 3.7% formaldehyde (Sigma) for 20 min on ice and permeabilized with Triton X-100 (0.25%) for 5 min at room temperature followed by ice-cold methanol for 10 min, incubated with the rabbit polyclonal anti-PCNA (diluted 1/50, clone Ab5 Calbiochem) for 45 min followed by biotinylated rabbit IgG, diluted 1:100 (Dako Cytomation) for 30 min, and then followed by streptavidin-coupled Alexa Fluor 555, diluted 1:200 (2 μg/ml, Molecular Probes) for 30 min. For HA or CRM1 detection, HeLa cells were fixed as described for PCNA labeling and were incubated with a rabbit polyclonal anti-HA (diluted 1:100, Roche Applied Science) or anti-CRM1 (Santa Cruz Biotechnology) antibody, respectively, followed by an Alexa Fluor 555-coupled goat-anti rabbit IgG (diluted 1:100, Molecular Probes). .. The analysis of the subcellular localization of the NES1-EGFP and NES2-EGFP and EGFP-NES1 and EGFP-NES2 in HeLa cells was performed 24 h after transfection.

    Protease Inhibitor:

    Article Title: Cell cycle-dependent ubiquitylation and destruction of NDE1 by CDK5-FBW7 regulates ciliary length
    Article Snippet: Plasmids were transfected into 293T cells using calcium phosphate method, as described (Kim et al , ). .. HEK293T, BALB/C 3T3, or RPE1-hTERT cells were lysed in 1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl at pH 7.5, 1 mM EGTA, 1 mM EDTA, 10% sucrose and protease inhibitor cocktail (Roche Applied Science), and phosphatase inhibitors 0.2 mM Na3 VO4 and 1 mM NaF at 4°C for 30 min. Antibodies against NDE1, (Proteintech), c-Myc, Flag (Sigma-Aldrich), CDK5 (Cell Signaling Technology), p35 (used to detect both p35 and p25) (C-19:sc-820, Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), mouse-FBW7 (Abnova), rabbit-FBW7 (A301-720A; A301-721A) (Bethyl Laboratories), rabbit-GLI2 (Cell Signaling), rabbit-Aurora A Kinase (Cell signaling), and α-tubulin (Sigma Aldrich) were used at a 1:1,000 dilution. .. Densitometric quantification of autoradiograms was analyzed by ImageJ software.

    Article Title: Varicella-Zoster Virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription
    Article Snippet: .. Whole cell lysates of VZV infected MeWo cells were prepared in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 0.1% Triton X-100 and protease inhibitor cocktail (Roche, Mannheim, GE, added per the manufacturer's instructions) and analyzed for IE62 and IE63 expression by immunoblot as previously described ( ) using rabbit antisera against full-length IE62 ( ) and IE63 ( ). .. Mouse monoclonal antibody against α-tubulin was obtained from Sigma-Aldrich (St. Louis, MO).

    Article Title: Boosting Replication and Penetration of Oncolytic Adenovirus by Paclitaxel Eradicate Peritoneal Metastasis of Gastric Cancer
    Article Snippet: The fluorescence-activated cell sorting (FACS) data were further analyzed using FlowJo Software (BD Biosciences) to calculate each fraction of cells in G1, S, and G2/M phases. .. Western Blot Analysis GCIY and KATOIII cells were seeded in a 100-mm dish at a density of 1 × 105 cells/dish and infected with OBP-401 at the indicated MOIs for 72 h. Cells were treated with PTX at the indicated doses for 24 h. Whole-cell lysates were prepared with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100) containing a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Mannheim, Germany). .. Proteins were electrophoresed on 8%–15% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK).

    Infection:

    Article Title: Varicella-Zoster Virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription
    Article Snippet: .. Whole cell lysates of VZV infected MeWo cells were prepared in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 0.1% Triton X-100 and protease inhibitor cocktail (Roche, Mannheim, GE, added per the manufacturer's instructions) and analyzed for IE62 and IE63 expression by immunoblot as previously described ( ) using rabbit antisera against full-length IE62 ( ) and IE63 ( ). .. Mouse monoclonal antibody against α-tubulin was obtained from Sigma-Aldrich (St. Louis, MO).

    Article Title: Boosting Replication and Penetration of Oncolytic Adenovirus by Paclitaxel Eradicate Peritoneal Metastasis of Gastric Cancer
    Article Snippet: The fluorescence-activated cell sorting (FACS) data were further analyzed using FlowJo Software (BD Biosciences) to calculate each fraction of cells in G1, S, and G2/M phases. .. Western Blot Analysis GCIY and KATOIII cells were seeded in a 100-mm dish at a density of 1 × 105 cells/dish and infected with OBP-401 at the indicated MOIs for 72 h. Cells were treated with PTX at the indicated doses for 24 h. Whole-cell lysates were prepared with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100) containing a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Mannheim, Germany). .. Proteins were electrophoresed on 8%–15% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK).

    Lysis:

    Article Title: Varicella-Zoster Virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription
    Article Snippet: .. Whole cell lysates of VZV infected MeWo cells were prepared in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 0.1% Triton X-100 and protease inhibitor cocktail (Roche, Mannheim, GE, added per the manufacturer's instructions) and analyzed for IE62 and IE63 expression by immunoblot as previously described ( ) using rabbit antisera against full-length IE62 ( ) and IE63 ( ). .. Mouse monoclonal antibody against α-tubulin was obtained from Sigma-Aldrich (St. Louis, MO).

    Article Title: Boosting Replication and Penetration of Oncolytic Adenovirus by Paclitaxel Eradicate Peritoneal Metastasis of Gastric Cancer
    Article Snippet: The fluorescence-activated cell sorting (FACS) data were further analyzed using FlowJo Software (BD Biosciences) to calculate each fraction of cells in G1, S, and G2/M phases. .. Western Blot Analysis GCIY and KATOIII cells were seeded in a 100-mm dish at a density of 1 × 105 cells/dish and infected with OBP-401 at the indicated MOIs for 72 h. Cells were treated with PTX at the indicated doses for 24 h. Whole-cell lysates were prepared with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100) containing a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Mannheim, Germany). .. Proteins were electrophoresed on 8%–15% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK).

    Expressing:

    Article Title: Varicella-Zoster Virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription
    Article Snippet: .. Whole cell lysates of VZV infected MeWo cells were prepared in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 0.1% Triton X-100 and protease inhibitor cocktail (Roche, Mannheim, GE, added per the manufacturer's instructions) and analyzed for IE62 and IE63 expression by immunoblot as previously described ( ) using rabbit antisera against full-length IE62 ( ) and IE63 ( ). .. Mouse monoclonal antibody against α-tubulin was obtained from Sigma-Aldrich (St. Louis, MO).

    Western Blot:

    Article Title: Boosting Replication and Penetration of Oncolytic Adenovirus by Paclitaxel Eradicate Peritoneal Metastasis of Gastric Cancer
    Article Snippet: The fluorescence-activated cell sorting (FACS) data were further analyzed using FlowJo Software (BD Biosciences) to calculate each fraction of cells in G1, S, and G2/M phases. .. Western Blot Analysis GCIY and KATOIII cells were seeded in a 100-mm dish at a density of 1 × 105 cells/dish and infected with OBP-401 at the indicated MOIs for 72 h. Cells were treated with PTX at the indicated doses for 24 h. Whole-cell lysates were prepared with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100) containing a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Mannheim, Germany). .. Proteins were electrophoresed on 8%–15% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK).

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    Roche triton x 100
    Effect of N-Ethylmaleimide (NEM) on the distribution of endogenous caspase-2 (C2) (Panels A–C) and FLAG-ERα (Panel D) between nuclear and extra-nuclear (cytosol or lysate) cell fractions. (A) Caspase-2 in cytosol and nuclei of cells without (−) or with (+) NEM (20 mM) pre-treatment for 10 min prior to cell fractionation using hypotonic buffer without detergent. (B) Caspase-2 in the lysate and nuclei of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. (C) Nuclear Caspase-2 in cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to lysis using the following conditions: 1) after lysis in cell culture plates (lysis buffer added directly to the plate wells), 2) cells in suspension collected in an Eppendorf tube were instantly frozen in dry ice/ethanol, transferred to ice bath and ice-cold lysis buffer with Triton X-100 was immediately added to the frozen pellets, 3) cells in suspension collected in an Eppendorf tube at room temperature and room temperature lysis buffer with 0.5% Triton X-100 was added to the pellets. (D). FLAG-tagged ERα in the lysate and nuclei of cells without (−) and with (+) NEM (20 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. The numbers on the right of the panels reflect the gel migration of the 40 kDa, 55 kDa, and 70 kDa protein markers. To ensure that our cell lysis procedure actually reflects nuclear and extra-nuclear (LYSATE) fractions, Western blotting studies examined for Histone H1.2 as a nuclear marker (E) and Procaspase-3 as an extra-nuclear marker (F)   [31] . Cell lysis was carried out using the conditions given in (B) above.
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    Effect of N-Ethylmaleimide (NEM) on the distribution of endogenous caspase-2 (C2) (Panels A–C) and FLAG-ERα (Panel D) between nuclear and extra-nuclear (cytosol or lysate) cell fractions. (A) Caspase-2 in cytosol and nuclei of cells without (−) or with (+) NEM (20 mM) pre-treatment for 10 min prior to cell fractionation using hypotonic buffer without detergent. (B) Caspase-2 in the lysate and nuclei of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. (C) Nuclear Caspase-2 in cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to lysis using the following conditions: 1) after lysis in cell culture plates (lysis buffer added directly to the plate wells), 2) cells in suspension collected in an Eppendorf tube were instantly frozen in dry ice/ethanol, transferred to ice bath and ice-cold lysis buffer with Triton X-100 was immediately added to the frozen pellets, 3) cells in suspension collected in an Eppendorf tube at room temperature and room temperature lysis buffer with 0.5% Triton X-100 was added to the pellets. (D). FLAG-tagged ERα in the lysate and nuclei of cells without (−) and with (+) NEM (20 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. The numbers on the right of the panels reflect the gel migration of the 40 kDa, 55 kDa, and 70 kDa protein markers. To ensure that our cell lysis procedure actually reflects nuclear and extra-nuclear (LYSATE) fractions, Western blotting studies examined for Histone H1.2 as a nuclear marker (E) and Procaspase-3 as an extra-nuclear marker (F)   [31] . Cell lysis was carried out using the conditions given in (B) above.

    Journal: PLoS ONE

    Article Title: A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli

    doi: 10.1371/journal.pone.0061085

    Figure Lengend Snippet: Effect of N-Ethylmaleimide (NEM) on the distribution of endogenous caspase-2 (C2) (Panels A–C) and FLAG-ERα (Panel D) between nuclear and extra-nuclear (cytosol or lysate) cell fractions. (A) Caspase-2 in cytosol and nuclei of cells without (−) or with (+) NEM (20 mM) pre-treatment for 10 min prior to cell fractionation using hypotonic buffer without detergent. (B) Caspase-2 in the lysate and nuclei of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. (C) Nuclear Caspase-2 in cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to lysis using the following conditions: 1) after lysis in cell culture plates (lysis buffer added directly to the plate wells), 2) cells in suspension collected in an Eppendorf tube were instantly frozen in dry ice/ethanol, transferred to ice bath and ice-cold lysis buffer with Triton X-100 was immediately added to the frozen pellets, 3) cells in suspension collected in an Eppendorf tube at room temperature and room temperature lysis buffer with 0.5% Triton X-100 was added to the pellets. (D). FLAG-tagged ERα in the lysate and nuclei of cells without (−) and with (+) NEM (20 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. The numbers on the right of the panels reflect the gel migration of the 40 kDa, 55 kDa, and 70 kDa protein markers. To ensure that our cell lysis procedure actually reflects nuclear and extra-nuclear (LYSATE) fractions, Western blotting studies examined for Histone H1.2 as a nuclear marker (E) and Procaspase-3 as an extra-nuclear marker (F) [31] . Cell lysis was carried out using the conditions given in (B) above.

    Article Snippet: In cells were disrupted in hypotonic buffer without Triton X-100 while in cells were lysed in the presence of 0.5% Triton X-100.

    Techniques: Cell Fractionation, Lysis, Cell Culture, Migration, Western Blot, Marker

    PSSA-2 requires the cytoplasmic tail for surface localisation. A. Schematic representation of PSSA-2. Predicted structural domains and signal peptide are indicated (not drawn to scale). AnTat 1.1 encodes a polypeptide of 436 amino acids, whereas the genome strain TREU 927/4 encodes a polypeptide of 425 amino acids. The sequence corresponding to the transmembrane domain is underlined; threonine residue T 305  is in boldface type and underlined. B. Immunoblot analysis of total lysates from procyclic forms of AnTat 1.1 stably transfected with plasmids encoding either an HA-tagged version of truncated PSSA-2 (ΔPSSA-2), lacking the cytoplasmic domain from residues 292–436, or full-length PSSA-2. Proteins were detected with an anti-HA antibody. 10 6  cell equivalents were loaded per lane. Markers are indicated on the left. C. Immunofluorescence analysis of HA-tagged full length PSSA-2 (top panel) and a ΔPSSA-2/GFP fusion protein (lower panel). Trypanosomes were fixed with formaldehyde and glutaraldehyde, permeabilized with Triton X-100 and stained with anti-GPEET, anti-HA or anti-BiP antibodies as indicated.

    Journal: PLoS ONE

    Article Title: PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei, Is Required for Efficient Maturation of Infection

    doi: 10.1371/journal.pone.0007074

    Figure Lengend Snippet: PSSA-2 requires the cytoplasmic tail for surface localisation. A. Schematic representation of PSSA-2. Predicted structural domains and signal peptide are indicated (not drawn to scale). AnTat 1.1 encodes a polypeptide of 436 amino acids, whereas the genome strain TREU 927/4 encodes a polypeptide of 425 amino acids. The sequence corresponding to the transmembrane domain is underlined; threonine residue T 305 is in boldface type and underlined. B. Immunoblot analysis of total lysates from procyclic forms of AnTat 1.1 stably transfected with plasmids encoding either an HA-tagged version of truncated PSSA-2 (ΔPSSA-2), lacking the cytoplasmic domain from residues 292–436, or full-length PSSA-2. Proteins were detected with an anti-HA antibody. 10 6 cell equivalents were loaded per lane. Markers are indicated on the left. C. Immunofluorescence analysis of HA-tagged full length PSSA-2 (top panel) and a ΔPSSA-2/GFP fusion protein (lower panel). Trypanosomes were fixed with formaldehyde and glutaraldehyde, permeabilized with Triton X-100 and stained with anti-GPEET, anti-HA or anti-BiP antibodies as indicated.

    Article Snippet: Cells were washed once in cold PBS and the pellet resuspended in 1 ml of Lysis Buffer (20 mM Tris-HCl pH 7.5, 2 mM EDTA, 2 mM EGTA, 1% (v/v) Triton X-100) supplemented with protease inhibitor cocktail (Roche Applied Science) according to manufacturer's instructions.

    Techniques: Sequencing, Stable Transfection, Transfection, Immunofluorescence, Staining

    Increased Pgp function (as observed in the uptake assays; see   Figures 2 ,   6 , and   7 ) is associated with the transition of Pgp from detergent (Lubrol) resistant membrane (DRM) domains to detergent soluble membrane domains. Doxycycline-induced hCMEC/D3-MDR1-EGFP cells were treated with MMC (1 µM) for 2 or 4 h and analyzed at the end of the 2 h-exposure period or 20 h after the 4 h-exposure period. Cell surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin. After solubilisation of these cells with 1% (w/v) of Lubrol WX, the lysates were centrifuged at 100,000× g for 45 min at 4°C. The DRMs (pellets) resuspended in 0.5 (w/v) DOC and 0.5% (w/v) Triton X-100 and the soluble fractions (supernatant) were subjected to Neutravidin beads to isolate cell surface proteins. These were then analyzed by Western blotting using antibodies against Pgp. The protein bands were analyzed by scanning densitometry. In “A” and “B”, Pgp bands in DRMs and supernatant are presented as percentage of the control in one representative experiment. Data in “C” and “D” are shown as means ± SEM of three experiments. Asterisks denote values that significantly differed (P

    Journal: PLoS ONE

    Article Title: Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

    doi: 10.1371/journal.pone.0088154

    Figure Lengend Snippet: Increased Pgp function (as observed in the uptake assays; see Figures 2 , 6 , and 7 ) is associated with the transition of Pgp from detergent (Lubrol) resistant membrane (DRM) domains to detergent soluble membrane domains. Doxycycline-induced hCMEC/D3-MDR1-EGFP cells were treated with MMC (1 µM) for 2 or 4 h and analyzed at the end of the 2 h-exposure period or 20 h after the 4 h-exposure period. Cell surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin. After solubilisation of these cells with 1% (w/v) of Lubrol WX, the lysates were centrifuged at 100,000× g for 45 min at 4°C. The DRMs (pellets) resuspended in 0.5 (w/v) DOC and 0.5% (w/v) Triton X-100 and the soluble fractions (supernatant) were subjected to Neutravidin beads to isolate cell surface proteins. These were then analyzed by Western blotting using antibodies against Pgp. The protein bands were analyzed by scanning densitometry. In “A” and “B”, Pgp bands in DRMs and supernatant are presented as percentage of the control in one representative experiment. Data in “C” and “D” are shown as means ± SEM of three experiments. Asterisks denote values that significantly differed (P

    Article Snippet: The cell pellets were washed with 5 ml Tris-buffered saline (TBS; 25 mM Tris, 0.15 M sodium chloride, pH 7.2) and centrifuged at 500× g for 5 min. After discarding the supernatants, cell pellets were resuspended in lysis buffer (25 mM Tris-HCl, 50 mM NaCl, 0.5% (w/v) DOC and 0.5% (w/v) Triton X-100) supplemented with complete protease inhibitor (Roche).

    Techniques: Western Blot

    Overexpressed human tau is secreted by Hela cells. (A) No tubulin was noted in M before and after overexpression of human tau whereas tubulin staining was detected in the cell lysate (Total lysis) prepared in 6 ml of lysis buffer for comparison with the 6 ml of medium used to maintain Hela cells after transfection (arrow). In M collected from Hela cells overexpressing tau that were partially lysed (Partial Lysis) for few seconds in a solution of 0.01% Triton X-100 to induce some damage at the plasma membrane, tubulin staining became detectable (asterisk). (B) Cleaved tau was detected in M and L (lower arrow) whereas full-length tau was only detected in L (upper arrow in Total lysis). Full-length tau became detectable in M when Hela cells were partially lysed (Partial lysis) with a solution of 0.01% Triton X-100 (upper arrow). (C) Hela cells overexpressing human tau were stained with Trypan blue before being fixed to evaluate the percentage of cell death. Blue cells (arrow) corresponded to dead cells that had taken up Trypan blue.

    Journal: PLoS ONE

    Article Title: Hyperphosphorylation and Cleavage at D421 Enhance Tau Secretion

    doi: 10.1371/journal.pone.0036873

    Figure Lengend Snippet: Overexpressed human tau is secreted by Hela cells. (A) No tubulin was noted in M before and after overexpression of human tau whereas tubulin staining was detected in the cell lysate (Total lysis) prepared in 6 ml of lysis buffer for comparison with the 6 ml of medium used to maintain Hela cells after transfection (arrow). In M collected from Hela cells overexpressing tau that were partially lysed (Partial Lysis) for few seconds in a solution of 0.01% Triton X-100 to induce some damage at the plasma membrane, tubulin staining became detectable (asterisk). (B) Cleaved tau was detected in M and L (lower arrow) whereas full-length tau was only detected in L (upper arrow in Total lysis). Full-length tau became detectable in M when Hela cells were partially lysed (Partial lysis) with a solution of 0.01% Triton X-100 (upper arrow). (C) Hela cells overexpressing human tau were stained with Trypan blue before being fixed to evaluate the percentage of cell death. Blue cells (arrow) corresponded to dead cells that had taken up Trypan blue.

    Article Snippet: The cells were then lysed in 6 ml of fresh culture medium supplemented with 0.1% Triton X-100 and protease inhibitor cocktail 1X (Complete EDTA-free from Roche Diagnostics, Indianapolis, IN, USA) and then incubated on ice for 10 min.

    Techniques: Over Expression, Staining, Lysis, Transfection