Structured Review

GE Healthcare esppα
Influence of buffer composition on the activity of <t>EspPα.</t> Relative activity is normalized to the maximal activity observed for 82.5 mM Mg 2+ . n = 8.
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1) Product Images from "Biochemical Characterization of the SPATE Members EspPα and EspI"

Article Title: Biochemical Characterization of the SPATE Members EspPα and EspI

Journal: Toxins

doi: 10.3390/toxins6092719

Influence of buffer composition on the activity of EspPα. Relative activity is normalized to the maximal activity observed for 82.5 mM Mg 2+ . n = 8.
Figure Legend Snippet: Influence of buffer composition on the activity of EspPα. Relative activity is normalized to the maximal activity observed for 82.5 mM Mg 2+ . n = 8.

Techniques Used: Activity Assay

Determination of the pH optimum of EspPα. Activity of EspPα was determined in the pH range from 5.5 to 9.1. The proteolytic activity is expressed relative to pH opt at ~7.4, n = 5.
Figure Legend Snippet: Determination of the pH optimum of EspPα. Activity of EspPα was determined in the pH range from 5.5 to 9.1. The proteolytic activity is expressed relative to pH opt at ~7.4, n = 5.

Techniques Used: Activity Assay

Temperature optimum and heat denaturation of EspPα ( a ) Relative activity of EspPα at varying incubation temperatures. Relative activity is normalized to T opt at ~40 °C, n = 8; ( b ) Effect of 30 min pre-incubation at elevated temperatures on the activity of EspPα at 37 °C. Pre-incubation temperatures are given in the x -axis and the relative activity was subsequently determined at 37 °C. The negative control was incubated for 30 min at 20 °C. Relative activity is normalized to the negative control, n = 8.
Figure Legend Snippet: Temperature optimum and heat denaturation of EspPα ( a ) Relative activity of EspPα at varying incubation temperatures. Relative activity is normalized to T opt at ~40 °C, n = 8; ( b ) Effect of 30 min pre-incubation at elevated temperatures on the activity of EspPα at 37 °C. Pre-incubation temperatures are given in the x -axis and the relative activity was subsequently determined at 37 °C. The negative control was incubated for 30 min at 20 °C. Relative activity is normalized to the negative control, n = 8.

Techniques Used: Activity Assay, Incubation, Negative Control

Purification of EspPα and EspI ( left ) SDS-PAGE of purified EspPα. * , EspPα autoproteolysis product; ( right ) SDS-PAGE of purified EspI. *, EspI autoproteolysis product. M = Molecular weight marker. Purity (including autoproteolysis products) of both samples was > 95% as determined by densitometrical analysis of SDS-PAGE gels.
Figure Legend Snippet: Purification of EspPα and EspI ( left ) SDS-PAGE of purified EspPα. * , EspPα autoproteolysis product; ( right ) SDS-PAGE of purified EspI. *, EspI autoproteolysis product. M = Molecular weight marker. Purity (including autoproteolysis products) of both samples was > 95% as determined by densitometrical analysis of SDS-PAGE gels.

Techniques Used: Purification, SDS Page, Molecular Weight, Marker

2) Product Images from "VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase"

Article Title: VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase

Journal: BMC Biology

doi: 10.1186/1741-7007-12-39

FAF1 interacts with VAPA and VAPB. (A) Flag-FAF1 immunoprecipitated from U2OS cells interacts with p97, VAPA and VAPB. (B) Flag-VAPA/B immunoprecipitated from U2OS cells interact with p97 and FAF1. (C) VAPB interaction with p97 is dependent on FAF1. U2OS cells were treated with the indicated siRNA oligos; luciferase (Luc) siRNA was used as a control. Flag-VAPB was immunoprecipitated and immunoblots of the immunoprecipitates (right) show that FAF1 depletion reduces the interaction with p97 whereas p97 depletion does not significantly affect the interaction with FAF1. (D) Endogenous VAPB interacts with p97 and FAF1 in mouse brain. Endogenous VAPB was immunoprecipitated from mouse brain extracts using Protein A-Sepharose (PAS) beads cross-linked to anti-VAPB antibodies. Uncoupled beads were used as a control. (E) Endogenous FAF1 interacts with VAPB in U2OS cells. The immunoprecipitation was performed using sheep anti-FAF1 antibody or sheep immunoglobulin G (IgG) as a control and PAS beads. (F) Indirect immunofluorescence of VAPB and wild-type (WT) Flag-FAF1. U2OS cells expressing Flag-FAF1 from a tetracycline-inducible promoter were grown in the presence of 200 ng/ml tetracycline for 24 hr and treated with 10 μM MG132 for 2 hr. Flag-FAF1 WT (red) co-localizes with VAPB (green) in a peri-nuclear area (enlarged window), suggesting an ER pattern. Scale bar is 10 μm. (G) VAPB levels and its interaction with Flag-FAF1 are not affected upon proteasome inhibition. Flag-FAF1 was immunoprecipitated from U2OS cells treated with 10 μM MG132 for 2 hr, 5 μM MG132 for 6 hr or left untreated (0 hr). Ubiquitinated proteins, p97 and VAPB were detected by immunoblotting in inputs (left) and immunoprecipitates (right). DAPI, 4',6-diamidino-2-phenylindole; IgG, immunoglobulin G; IP, immunoprecipitate; Luc, luciferase; WT, wild type.
Figure Legend Snippet: FAF1 interacts with VAPA and VAPB. (A) Flag-FAF1 immunoprecipitated from U2OS cells interacts with p97, VAPA and VAPB. (B) Flag-VAPA/B immunoprecipitated from U2OS cells interact with p97 and FAF1. (C) VAPB interaction with p97 is dependent on FAF1. U2OS cells were treated with the indicated siRNA oligos; luciferase (Luc) siRNA was used as a control. Flag-VAPB was immunoprecipitated and immunoblots of the immunoprecipitates (right) show that FAF1 depletion reduces the interaction with p97 whereas p97 depletion does not significantly affect the interaction with FAF1. (D) Endogenous VAPB interacts with p97 and FAF1 in mouse brain. Endogenous VAPB was immunoprecipitated from mouse brain extracts using Protein A-Sepharose (PAS) beads cross-linked to anti-VAPB antibodies. Uncoupled beads were used as a control. (E) Endogenous FAF1 interacts with VAPB in U2OS cells. The immunoprecipitation was performed using sheep anti-FAF1 antibody or sheep immunoglobulin G (IgG) as a control and PAS beads. (F) Indirect immunofluorescence of VAPB and wild-type (WT) Flag-FAF1. U2OS cells expressing Flag-FAF1 from a tetracycline-inducible promoter were grown in the presence of 200 ng/ml tetracycline for 24 hr and treated with 10 μM MG132 for 2 hr. Flag-FAF1 WT (red) co-localizes with VAPB (green) in a peri-nuclear area (enlarged window), suggesting an ER pattern. Scale bar is 10 μm. (G) VAPB levels and its interaction with Flag-FAF1 are not affected upon proteasome inhibition. Flag-FAF1 was immunoprecipitated from U2OS cells treated with 10 μM MG132 for 2 hr, 5 μM MG132 for 6 hr or left untreated (0 hr). Ubiquitinated proteins, p97 and VAPB were detected by immunoblotting in inputs (left) and immunoprecipitates (right). DAPI, 4',6-diamidino-2-phenylindole; IgG, immunoglobulin G; IP, immunoprecipitate; Luc, luciferase; WT, wild type.

Techniques Used: Immunoprecipitation, Luciferase, Western Blot, Immunofluorescence, Expressing, Inhibition

3) Product Images from "Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain"

Article Title: Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain

Journal:

doi: 10.1016/j.jmb.2007.10.060

Reduction of TA0289 by dithionite and reoxidation by air. 19 µM TA0289 in 50 mM Hepes-Na + (pH 7.5), 0.2 M NaCl, and 10% glycerol (trace a ) was scanned. A 50 mM stock of anaerobic sodium dithionite was then diluted 100-fold into the solution (to
Figure Legend Snippet: Reduction of TA0289 by dithionite and reoxidation by air. 19 µM TA0289 in 50 mM Hepes-Na + (pH 7.5), 0.2 M NaCl, and 10% glycerol (trace a ) was scanned. A 50 mM stock of anaerobic sodium dithionite was then diluted 100-fold into the solution (to

Techniques Used:

Anaerobic reduction of TA0289 by the E. coli NorW in the presence of NADH. A 24 µM anaerobic sample of TA0289 in a solution containing 50 mM Hepes-Na + (pH 7.5), 0.2 M NaCl, 10% glycerol and 1 µM NorW was scanned (trace a ). Reduction of
Figure Legend Snippet: Anaerobic reduction of TA0289 by the E. coli NorW in the presence of NADH. A 24 µM anaerobic sample of TA0289 in a solution containing 50 mM Hepes-Na + (pH 7.5), 0.2 M NaCl, 10% glycerol and 1 µM NorW was scanned (trace a ). Reduction of

Techniques Used:

Reduction of cytochrome C by TA0289 in the presence of NorW and NADH. A 200 mg ml −1 sample of cytochrome C in 50 mM Hepes-Na (pH 7.5), 0.2 M NaCl, and 10% glycerol in the presence of 1 µM NorW and 1.9 µM TA0289 was scanned (trace
Figure Legend Snippet: Reduction of cytochrome C by TA0289 in the presence of NorW and NADH. A 200 mg ml −1 sample of cytochrome C in 50 mM Hepes-Na (pH 7.5), 0.2 M NaCl, and 10% glycerol in the presence of 1 µM NorW and 1.9 µM TA0289 was scanned (trace

Techniques Used:

4) Product Images from "Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞"

Article Title: Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞

Journal: Molecular Pharmacology

doi: 10.1124/mol.109.062539

MAM accommodates DRMs associated with Sig-1R. A, purification of MAM and microsomal fractions from CHO cells. Nuclear (P1), mitochondrial (Mito), MAM, microsomal (P3), and cytosolic (Cyt) fractions were prepared by differential centrifugation combined with a Percoll gradient fractionation. Five micrograms of proteins was applied to SDS-PAGE, followed by immunoblotting. COX, cytochrome c oxidase subunit I. Asterisks indicate ER chaperones involved in vesicular transport. Graphs represent fraction distributions of proteins in which the sum of five fractions was taken as 100% for each protein. B, Sig-1R-associated DRMs in the MAM. MAM and microsomes (P3) (25 μg of total proteins in each) were extracted by 0.5% Tx (Tx-100) or Triton X-114 at 4°C. DRMs and detergent-soluble supernatant (S) were prepared by differential centrifugations. The numbers represent the average of optical density (O.D.) measured in each protein band ( n = 3). C, silver staining for total proteins associated with DRMs in MAM and microsomal fractions. MAM and microsomes (25 μg of total proteins in each) were extracted in 0.5% Tx at 4°C, and DRMs and soluble supernatants (S) were prepared. Proteins were visualized by 13% SDS-PAGE, followed by silver staining. The numbers represent the average of O.D. measured in each lane ( n = 3). MW, molecular weight of standard proteins. D, lipid contents in MAM and microsomal fractions. Lipids were extracted and analyzed by HPTLC. Cholesterol (Chol) was detected using a ferric chloride spray; GlcCer using a diphenylamine-aniline spray. Lipids in the second panel were visualized under UV light after an ANS spray. In the lipid overlay assay for ceramides (Cer, bottom), ceramides extracted from HPTLC plates were immobilized on a nitrocellulose membrane followed by immunoblotting with anti-ceramide antibodies. SM, sphingomyelin.
Figure Legend Snippet: MAM accommodates DRMs associated with Sig-1R. A, purification of MAM and microsomal fractions from CHO cells. Nuclear (P1), mitochondrial (Mito), MAM, microsomal (P3), and cytosolic (Cyt) fractions were prepared by differential centrifugation combined with a Percoll gradient fractionation. Five micrograms of proteins was applied to SDS-PAGE, followed by immunoblotting. COX, cytochrome c oxidase subunit I. Asterisks indicate ER chaperones involved in vesicular transport. Graphs represent fraction distributions of proteins in which the sum of five fractions was taken as 100% for each protein. B, Sig-1R-associated DRMs in the MAM. MAM and microsomes (P3) (25 μg of total proteins in each) were extracted by 0.5% Tx (Tx-100) or Triton X-114 at 4°C. DRMs and detergent-soluble supernatant (S) were prepared by differential centrifugations. The numbers represent the average of optical density (O.D.) measured in each protein band ( n = 3). C, silver staining for total proteins associated with DRMs in MAM and microsomal fractions. MAM and microsomes (25 μg of total proteins in each) were extracted in 0.5% Tx at 4°C, and DRMs and soluble supernatants (S) were prepared. Proteins were visualized by 13% SDS-PAGE, followed by silver staining. The numbers represent the average of O.D. measured in each lane ( n = 3). MW, molecular weight of standard proteins. D, lipid contents in MAM and microsomal fractions. Lipids were extracted and analyzed by HPTLC. Cholesterol (Chol) was detected using a ferric chloride spray; GlcCer using a diphenylamine-aniline spray. Lipids in the second panel were visualized under UV light after an ANS spray. In the lipid overlay assay for ceramides (Cer, bottom), ceramides extracted from HPTLC plates were immobilized on a nitrocellulose membrane followed by immunoblotting with anti-ceramide antibodies. SM, sphingomyelin.

Techniques Used: Purification, Centrifugation, Fractionation, SDS Page, Silver Staining, Molecular Weight, High Performance Thin Layer Chromatography, Overlay Assay

5) Product Images from "Sumoylation inhibits ?-synuclein aggregation and toxicity"

Article Title: Sumoylation inhibits ?-synuclein aggregation and toxicity

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201010117

In vitro fibril formation of sumoylated α-synuclein. (A) Coomassie staining of α-synuclein sumoylated to 100% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (B) Fibrillization kinetics of α-synuclein sumoylated to 100% (70 µM; closed circles) and control condition (70 µM of nonmodified α-synuclein + 70 µM of free SUMO1; open circles). (C) Nonamyloidogenic amorphous oligomers formed by α-synuclein sumoylated to 100%. (D) Mature fibrils formed by control nonmodified α-synuclein in the presence of 70 µM of free SUMO1. (E) Coomassie staining of α-synuclein sumoylated to 50% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (F) Fibrillization kinetics of α-synuclein sumoylated to 50% (35 µM sumoylated α-synuclein + 35 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 35 µM of free SUMO1; open circles). (G) Nonamyloidogenic amorphous material formed by α-synuclein sumoylated to 50%. (H) Mature fibrils formed by control nonmodified α-synuclein in the presence of 35 µM of free SUMO1. (I) Coomassie staining of α-synuclein sumoylated to 10% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (J) Fibrillization kinetics of α-synuclein sumoylated to 10% (7 µM sumoylated α-synuclein + 63 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 7 µM of free SUMO1; open circles). (K) Mature fibrils formed by α-synuclein sumoylated to 10%. (L) Mature fibrils formed by control nonmodified α-synuclein in the presence of 7 µM of free SUMO1. (C, D, G, H, K, and L) TEM of aggregation samples after 146 h of incubation in 50 mM Hepes and 100 mM NaCl, pH 7.4, at 37°C with constant stirring. ThT, Thioflavin T. Bars, 200 nm.
Figure Legend Snippet: In vitro fibril formation of sumoylated α-synuclein. (A) Coomassie staining of α-synuclein sumoylated to 100% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (B) Fibrillization kinetics of α-synuclein sumoylated to 100% (70 µM; closed circles) and control condition (70 µM of nonmodified α-synuclein + 70 µM of free SUMO1; open circles). (C) Nonamyloidogenic amorphous oligomers formed by α-synuclein sumoylated to 100%. (D) Mature fibrils formed by control nonmodified α-synuclein in the presence of 70 µM of free SUMO1. (E) Coomassie staining of α-synuclein sumoylated to 50% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (F) Fibrillization kinetics of α-synuclein sumoylated to 50% (35 µM sumoylated α-synuclein + 35 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 35 µM of free SUMO1; open circles). (G) Nonamyloidogenic amorphous material formed by α-synuclein sumoylated to 50%. (H) Mature fibrils formed by control nonmodified α-synuclein in the presence of 35 µM of free SUMO1. (I) Coomassie staining of α-synuclein sumoylated to 10% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (J) Fibrillization kinetics of α-synuclein sumoylated to 10% (7 µM sumoylated α-synuclein + 63 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 7 µM of free SUMO1; open circles). (K) Mature fibrils formed by α-synuclein sumoylated to 10%. (L) Mature fibrils formed by control nonmodified α-synuclein in the presence of 7 µM of free SUMO1. (C, D, G, H, K, and L) TEM of aggregation samples after 146 h of incubation in 50 mM Hepes and 100 mM NaCl, pH 7.4, at 37°C with constant stirring. ThT, Thioflavin T. Bars, 200 nm.

Techniques Used: In Vitro, Staining, Transmission Electron Microscopy, Incubation

6) Product Images from "Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity"

Article Title: Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049048

Kinetic analysis of PI-1 SrtC1 and SrtC2 wild-type and mutants. Triplicate data sets for each experiment were used to calculate the steady-state velocity at different PI-1 peptides concentrations for each enzyme and were expressed as initial rates (µM/s) versus concentration of substrate. SrtC1 (top) and SrtC2 (bottom) enzymes carrying the mutation Y92A and F86A (SrtC1 Y92A and SrtC2 F86A ) and the deletion of the entire N-terminal region (SrtC1 ΔNT and SrtC2 ΔNT ) were analyzed in comparison with wild-type enzymes by FRET assays at various concentrations of three different PI-1 peptides ( Table 2 ). The reactions containing 25 µM of enzyme and 2–128 µM of fluorescent peptide were performed at 37°C in 20 mM HEPES pH 7.5, 100 mM NaCl and 1 mM DTT.
Figure Legend Snippet: Kinetic analysis of PI-1 SrtC1 and SrtC2 wild-type and mutants. Triplicate data sets for each experiment were used to calculate the steady-state velocity at different PI-1 peptides concentrations for each enzyme and were expressed as initial rates (µM/s) versus concentration of substrate. SrtC1 (top) and SrtC2 (bottom) enzymes carrying the mutation Y92A and F86A (SrtC1 Y92A and SrtC2 F86A ) and the deletion of the entire N-terminal region (SrtC1 ΔNT and SrtC2 ΔNT ) were analyzed in comparison with wild-type enzymes by FRET assays at various concentrations of three different PI-1 peptides ( Table 2 ). The reactions containing 25 µM of enzyme and 2–128 µM of fluorescent peptide were performed at 37°C in 20 mM HEPES pH 7.5, 100 mM NaCl and 1 mM DTT.

Techniques Used: Concentration Assay, Mutagenesis

FRET assay with PI-1 and PI-2a peptides for substrate specificity analysis of PI-1 SrtC1 and SrtC2. (A) The reaction solutions contained 128 µM of PI-1 fluorescent peptides and 25 µM of SrtC1-TM (left panel) or SrtC2-TM (right panel). The reactions were performed at 37°C in the assay buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl and 1 mM DTT. Fluorescence emission was monitored every 10 minutes and an increase in fluorescence intensity was observed in the presence of BP, AP1 and AP2 peptides mimicking the LPXTG motif of PI-1 pilus proteins. (B) Reactions were performed with PI-2a peptides and 25 µM of SrtC1-TM in the same conditions described above. (C) In vivo substrate specificity analysis. Immunoblots of total protein extracts from GBS 515 (containing SrtC1 and SrtC2 of pilus island 2a) and JM9130013 (containing SrtC1 and SrtC2 of pilus islands 1 and 2b) wild-type and complemented strains with plasmids expressing the backbone proteins of PI-1 (BP-1) and PI-2a (BP-2a), respectively. The nitrocellulose membranes were probed with antisera specific for BP-1 and BP-2a.
Figure Legend Snippet: FRET assay with PI-1 and PI-2a peptides for substrate specificity analysis of PI-1 SrtC1 and SrtC2. (A) The reaction solutions contained 128 µM of PI-1 fluorescent peptides and 25 µM of SrtC1-TM (left panel) or SrtC2-TM (right panel). The reactions were performed at 37°C in the assay buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl and 1 mM DTT. Fluorescence emission was monitored every 10 minutes and an increase in fluorescence intensity was observed in the presence of BP, AP1 and AP2 peptides mimicking the LPXTG motif of PI-1 pilus proteins. (B) Reactions were performed with PI-2a peptides and 25 µM of SrtC1-TM in the same conditions described above. (C) In vivo substrate specificity analysis. Immunoblots of total protein extracts from GBS 515 (containing SrtC1 and SrtC2 of pilus island 2a) and JM9130013 (containing SrtC1 and SrtC2 of pilus islands 1 and 2b) wild-type and complemented strains with plasmids expressing the backbone proteins of PI-1 (BP-1) and PI-2a (BP-2a), respectively. The nitrocellulose membranes were probed with antisera specific for BP-1 and BP-2a.

Techniques Used: Fluorescence, In Vivo, Western Blot, Expressing

7) Product Images from "Gamma-secretase activating protein, a therapeutic target for Alzheimer's disease"

Article Title: Gamma-secretase activating protein, a therapeutic target for Alzheimer's disease

Journal: Nature

doi: 10.1038/nature09325

gSAP regulates Aβ production but does not influence Notch cleavage a: siRNA-mediated knockdown of gSAP in N2a cells overexpressing APP695 lowered Aβ production. The Aβ-lowering effects of imatinib and of siRNA were not additive (mean ±s.d.; ** p
Figure Legend Snippet: gSAP regulates Aβ production but does not influence Notch cleavage a: siRNA-mediated knockdown of gSAP in N2a cells overexpressing APP695 lowered Aβ production. The Aβ-lowering effects of imatinib and of siRNA were not additive (mean ±s.d.; ** p

Techniques Used:

Identification of gSAP as an imatinib target a: A PS1-associated 16 kDa protein is labeled by a photoactivatable imatinib derivative. Left panel: photolysis of 125 I-G01 with membrane preparations. Middle panel: photolysis of 3 H-G01 with intact HEK293 cells. Right panel: PS1-CTF migrated with a slower mobility than the labeled 16 kDa band and was not labeled by G01. Labeling specificity was confirmed by competition with unlabeled imatinib. b: Solubilized endogenous γ-secretase components from HEK293 cells were bound to immobilized biotin-imatinib (left panel). Among the proteins bound to biotin-imatinib, a ~ 16 kDa band was detected by silver staining and was identified as the C-terminal domain of gSAP (right panel, arrow and label “gSAP”). Biotin-coated beads and an inactive biotin-imatinib derivative (see supplementary Fig. 3 ) served as controls. c: Endogenous gSAP in N2a cells was synthesized as a full length 98 kDa-precursor protein and rapidly processed into a 16 kDa C-terminal fragment. Under steady-state conditions, the predominant cellular form of gSAP was 16 kDa. d: Endogenous gSAP-16K was specifically labeled by 3 H-G01 in neuroblastoma cells. e: After gSAP siRNA knockdown in N2a cells, immobilized biotin-imatinib no longer captured PS1.
Figure Legend Snippet: Identification of gSAP as an imatinib target a: A PS1-associated 16 kDa protein is labeled by a photoactivatable imatinib derivative. Left panel: photolysis of 125 I-G01 with membrane preparations. Middle panel: photolysis of 3 H-G01 with intact HEK293 cells. Right panel: PS1-CTF migrated with a slower mobility than the labeled 16 kDa band and was not labeled by G01. Labeling specificity was confirmed by competition with unlabeled imatinib. b: Solubilized endogenous γ-secretase components from HEK293 cells were bound to immobilized biotin-imatinib (left panel). Among the proteins bound to biotin-imatinib, a ~ 16 kDa band was detected by silver staining and was identified as the C-terminal domain of gSAP (right panel, arrow and label “gSAP”). Biotin-coated beads and an inactive biotin-imatinib derivative (see supplementary Fig. 3 ) served as controls. c: Endogenous gSAP in N2a cells was synthesized as a full length 98 kDa-precursor protein and rapidly processed into a 16 kDa C-terminal fragment. Under steady-state conditions, the predominant cellular form of gSAP was 16 kDa. d: Endogenous gSAP-16K was specifically labeled by 3 H-G01 in neuroblastoma cells. e: After gSAP siRNA knockdown in N2a cells, immobilized biotin-imatinib no longer captured PS1.

Techniques Used: Labeling, Silver Staining, Synthesized

gSAP interacts with γ-secretase and APP-CTF but not with Notch a: Endogenous gSAP-16K in solubilized membrane preparations from N2a cells co-migrated with γ-secretase components during gel filtration (void volume: fraction 6). b: Immunoprecipitation of endogenous gSAP from N2a cells resulted in co-immunoprecipitation of γ-secretase components. c: Endogenous gSAP-16K and γ-secretase components are highly enriched by an immobilized γ-secretase transition state analogue (GSI beads). d: In HEK293 cells, gSAP-16K and APP-CTF, but not NotchΔE, co-immunoprecipitated. e: Imatinib treatment reduced the co-immunoprecipitation of APP-CTF and gSAP in a concentration-dependent manner. An inactive imatinib derivative (IC200001, see supplementary Fig. 3 ) served as a negative control. f: In HEK293 cells, APP-CTF without the cytoplasmic domain (APPε-CTF) did not co-immunoprecipitate with gSAP-16K (upper panel); γ-cleavage of APPε-CTF was not stimulated by gSAP-16K in an in vitro assay (lower panel).
Figure Legend Snippet: gSAP interacts with γ-secretase and APP-CTF but not with Notch a: Endogenous gSAP-16K in solubilized membrane preparations from N2a cells co-migrated with γ-secretase components during gel filtration (void volume: fraction 6). b: Immunoprecipitation of endogenous gSAP from N2a cells resulted in co-immunoprecipitation of γ-secretase components. c: Endogenous gSAP-16K and γ-secretase components are highly enriched by an immobilized γ-secretase transition state analogue (GSI beads). d: In HEK293 cells, gSAP-16K and APP-CTF, but not NotchΔE, co-immunoprecipitated. e: Imatinib treatment reduced the co-immunoprecipitation of APP-CTF and gSAP in a concentration-dependent manner. An inactive imatinib derivative (IC200001, see supplementary Fig. 3 ) served as a negative control. f: In HEK293 cells, APP-CTF without the cytoplasmic domain (APPε-CTF) did not co-immunoprecipitate with gSAP-16K (upper panel); γ-cleavage of APPε-CTF was not stimulated by gSAP-16K in an in vitro assay (lower panel).

Techniques Used: Filtration, Immunoprecipitation, Concentration Assay, Negative Control, In Vitro

8) Product Images from "Molecular mechanism of APC/C activation by mitotic phosphorylation"

Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

Journal: Nature

doi: 10.1038/nature17973

Conformational changes of the APC/C between the inactive apo and the active ternary states and domain and sequence analysis of Cdc20. a, b , Subunits that undergo conformational changes upon coactivator and substrate binding are highlighted in their ternary state and coloured as in , while the corresponding proteins in the inactive apo state are in lighter shades. In the active conformation, the platform subdomain containing subunits Apc1, Apc4 and Apc5 is shifted upward, inducing a large movement of the catalytic module to enable E2 access. c , Domain organization of Cdc20. d , Sequence alignment of Cdc20 NTD and Cdh1 NTD with α-helices represented as cylinders (purple and grey for Cdc20 NTD and Cdh1 NTD , respectively) underneath the sequences and the C box and KILR/KLLR motif highlighted. Fig. 1
Figure Legend Snippet: Conformational changes of the APC/C between the inactive apo and the active ternary states and domain and sequence analysis of Cdc20. a, b , Subunits that undergo conformational changes upon coactivator and substrate binding are highlighted in their ternary state and coloured as in , while the corresponding proteins in the inactive apo state are in lighter shades. In the active conformation, the platform subdomain containing subunits Apc1, Apc4 and Apc5 is shifted upward, inducing a large movement of the catalytic module to enable E2 access. c , Domain organization of Cdc20. d , Sequence alignment of Cdc20 NTD and Cdh1 NTD with α-helices represented as cylinders (purple and grey for Cdc20 NTD and Cdh1 NTD , respectively) underneath the sequences and the C box and KILR/KLLR motif highlighted. Fig. 1

Techniques Used: Sequencing, Binding Assay, Cytotoxicity Assay

Comparison of Cdc20 and Cdh1 association to the APC/C. a , The catalytic module (Apc2-Apc11) of the APC/C Cdc20-Hsl1 complex is flexible and almost no density accounting for Apc11 (pink, modelled based on the structure of APC/C Cdh1-Emi1 could be observed. b , The WD40 domain of Cdc20 (purple) occupies a similar position as Cdh1 WD40 (grey), but it is displaced from the APC/C by as much as 10 Å. c, d , EM density for Cdc20 C box allowed for ab initio model building and the C-box interaction with Apc8B (cyan) is well conserved between the two coactivators. e , Both Cdc20 IR and Cdh1 IR associates with Apc3A (orange), although the EM density for Cdc20 IR is much weaker (not shown) and the C-terminal α-helix in Cdh1 IR is absent.
Figure Legend Snippet: Comparison of Cdc20 and Cdh1 association to the APC/C. a , The catalytic module (Apc2-Apc11) of the APC/C Cdc20-Hsl1 complex is flexible and almost no density accounting for Apc11 (pink, modelled based on the structure of APC/C Cdh1-Emi1 could be observed. b , The WD40 domain of Cdc20 (purple) occupies a similar position as Cdh1 WD40 (grey), but it is displaced from the APC/C by as much as 10 Å. c, d , EM density for Cdc20 C box allowed for ab initio model building and the C-box interaction with Apc8B (cyan) is well conserved between the two coactivators. e , Both Cdc20 IR and Cdh1 IR associates with Apc3A (orange), although the EM density for Cdc20 IR is much weaker (not shown) and the C-terminal α-helix in Cdh1 IR is absent.

Techniques Used:

The Apc1 AI segment binds to the C-box binding site and mimics the Cdc20 C box . a , Cdk2-cyclin A2-Cks2 was used for ubiquitination assays. In vitro phosphorylated APC/C (both Cdk2-cyclin A3-Cks2 and Plk1) can be activated by Cdc20 (lanes 1-5). Deletion of the Apc1 300s loop activated the APC/C without phosphorylation (lanes 6-7), and kinase treatment of APC/C ΔApc1-300s does not enhance APC/C activity. The APC/C ΔApc3-loop mutant showed similar activity as unphosphorylated APC/C (lanes 10-11 vs 2-3 and 4-5), but had reduced activation by phosphorylation. Nevertheless, deletion of both Apc1 300s and Apc3 loops (APC/C ΔApc1-300s Apc3-loop ) restored activity to that of WT phosphorylated APC/C and unphosphorylated APC/C ΔApc1-300s (lanes 14-17). b , Identification of the Apc1 AI segment occupying the C-box binding site by assessing the inhibitory effect of eight peptides spanning the Apc1 300s loop. A single peptide (peptide 7, residues 361-380) suppressed the activity of APC/C ΔApc1-300s (lane 9), indicating that this peptide blocks the C-box binding site. A control with WT unphosphorylated APC/C ( unp. APC/C WT ) is in lane 11. c , The Apc1 AI segment (peptide 7, residues 361-380) shares sequence similarity with Cdc20 C box . A model for the AI segment (green) was fitted into the EM density of the apo unphosphorylated APC/C map (grey). Arg368 overlaps with the crucial Arg78 of Cdc20 C box (purple, right panel). The flanking serines shown to be phosphorylated are highlighted as red spheres. Ser377 is outside the observed EM density. d , Mutation of a single Arg368 residue (APC/C Apc1-R368E ) or mutating its four neighbouring serine residues (Ser364, Ser372, Ser373, Ser377) to glutamates (APC/C Apc1-4S/E ) activated the APC/C without phosphorylation. 30 nM Cdc20 was used for assay in a and 20 nM Cdc20 for assays in b and d . Experiments in a and d were replicated three times and in b for gel source data.
Figure Legend Snippet: The Apc1 AI segment binds to the C-box binding site and mimics the Cdc20 C box . a , Cdk2-cyclin A2-Cks2 was used for ubiquitination assays. In vitro phosphorylated APC/C (both Cdk2-cyclin A3-Cks2 and Plk1) can be activated by Cdc20 (lanes 1-5). Deletion of the Apc1 300s loop activated the APC/C without phosphorylation (lanes 6-7), and kinase treatment of APC/C ΔApc1-300s does not enhance APC/C activity. The APC/C ΔApc3-loop mutant showed similar activity as unphosphorylated APC/C (lanes 10-11 vs 2-3 and 4-5), but had reduced activation by phosphorylation. Nevertheless, deletion of both Apc1 300s and Apc3 loops (APC/C ΔApc1-300s Apc3-loop ) restored activity to that of WT phosphorylated APC/C and unphosphorylated APC/C ΔApc1-300s (lanes 14-17). b , Identification of the Apc1 AI segment occupying the C-box binding site by assessing the inhibitory effect of eight peptides spanning the Apc1 300s loop. A single peptide (peptide 7, residues 361-380) suppressed the activity of APC/C ΔApc1-300s (lane 9), indicating that this peptide blocks the C-box binding site. A control with WT unphosphorylated APC/C ( unp. APC/C WT ) is in lane 11. c , The Apc1 AI segment (peptide 7, residues 361-380) shares sequence similarity with Cdc20 C box . A model for the AI segment (green) was fitted into the EM density of the apo unphosphorylated APC/C map (grey). Arg368 overlaps with the crucial Arg78 of Cdc20 C box (purple, right panel). The flanking serines shown to be phosphorylated are highlighted as red spheres. Ser377 is outside the observed EM density. d , Mutation of a single Arg368 residue (APC/C Apc1-R368E ) or mutating its four neighbouring serine residues (Ser364, Ser372, Ser373, Ser377) to glutamates (APC/C Apc1-4S/E ) activated the APC/C without phosphorylation. 30 nM Cdc20 was used for assay in a and 20 nM Cdc20 for assays in b and d . Experiments in a and d were replicated three times and in b for gel source data.

Techniques Used: Binding Assay, In Vitro, Activity Assay, Mutagenesis, Activation Assay, Sequencing

Three-dimensional classification of APC/C Cdc20-Hsl . The initial particles after 2-dimensional classification were divided into six classes by 3-dimensional classification module using RELION. The resultant classes were grouped into four categories: (i) 9.0% in the active ternary state with coactivator and substrate bound; (ii) 11.3% in a hybrid state with coactivator bound, but the APC/C in the inactive conformation; (iii) 71.6% in the inactive apo state; (iv) 8.1% has poorer reconstruction due to some bad particles. The first class in the active ternary state containing 179,660 particles was used for 3-dimensional refinement and movie correction to obtain the final reconstruction at 3.9 Å.
Figure Legend Snippet: Three-dimensional classification of APC/C Cdc20-Hsl . The initial particles after 2-dimensional classification were divided into six classes by 3-dimensional classification module using RELION. The resultant classes were grouped into four categories: (i) 9.0% in the active ternary state with coactivator and substrate bound; (ii) 11.3% in a hybrid state with coactivator bound, but the APC/C in the inactive conformation; (iii) 71.6% in the inactive apo state; (iv) 8.1% has poorer reconstruction due to some bad particles. The first class in the active ternary state containing 179,660 particles was used for 3-dimensional refinement and movie correction to obtain the final reconstruction at 3.9 Å.

Techniques Used:

Preparations and EM images of different APC/C samples used for structural studies. a , Recombinant human APC/C was in vitro phosphorylated using Cdk2-cyclin A3, Cdk2-cyclin A3-Cks2 or Plk1 alone or with both Cdk2-cyclin A3-Cks2 and Plk1. The phosphorylated APC/C samples are shown on SDS-PAGE. b , In vitro phosphorylated recombinant human APC/C can be fully activated by Cdc20 to ubiquitylate a native substrate Cdk2-cyclin A2-Cks2 when both kinases were added (lanes 9, 10). Without Cks2 (lanes 3, 4) or with Plk1 alone (lanes 7, 8) no activation of the APC/C could be observed, whereas treating with Cdk2-cyclin A3-Cks2 alone (lanes 5, 6) resulted in its partial activation. A time course was recorded at 15 and 30 min and 20 nM of Cdc20 was used. This experiment was replicated three times. Anti-Apc3 antibodies (BD Bioscience, cat. code: 610454) were used as a loading control. c , Purified APC/C WT and mutant samples with and without kinase treatment (both Cdk2-cyclin A3-Cks2 and Plk1). Upon deletion of the Apc3 loop, no association of the Cdk2-cyclin A3-Cks2 kinase to the APC/C could be observed (lanes 6 and 8). d , Purified APC/C Cdc20-Hsl1 ternary complex on SDS-PAGE. e , A typical cryo-EM micrograph of APC/C Cdc20-Hsl1 representative of 15,582 micrographs. f , Gallery of two-dimensional averages of APC/C Cdc20-Hsl1 showing different views; representative of 100 two-dimensional averages. g for gel source data.
Figure Legend Snippet: Preparations and EM images of different APC/C samples used for structural studies. a , Recombinant human APC/C was in vitro phosphorylated using Cdk2-cyclin A3, Cdk2-cyclin A3-Cks2 or Plk1 alone or with both Cdk2-cyclin A3-Cks2 and Plk1. The phosphorylated APC/C samples are shown on SDS-PAGE. b , In vitro phosphorylated recombinant human APC/C can be fully activated by Cdc20 to ubiquitylate a native substrate Cdk2-cyclin A2-Cks2 when both kinases were added (lanes 9, 10). Without Cks2 (lanes 3, 4) or with Plk1 alone (lanes 7, 8) no activation of the APC/C could be observed, whereas treating with Cdk2-cyclin A3-Cks2 alone (lanes 5, 6) resulted in its partial activation. A time course was recorded at 15 and 30 min and 20 nM of Cdc20 was used. This experiment was replicated three times. Anti-Apc3 antibodies (BD Bioscience, cat. code: 610454) were used as a loading control. c , Purified APC/C WT and mutant samples with and without kinase treatment (both Cdk2-cyclin A3-Cks2 and Plk1). Upon deletion of the Apc3 loop, no association of the Cdk2-cyclin A3-Cks2 kinase to the APC/C could be observed (lanes 6 and 8). d , Purified APC/C Cdc20-Hsl1 ternary complex on SDS-PAGE. e , A typical cryo-EM micrograph of APC/C Cdc20-Hsl1 representative of 15,582 micrographs. f , Gallery of two-dimensional averages of APC/C Cdc20-Hsl1 showing different views; representative of 100 two-dimensional averages. g for gel source data.

Techniques Used: Recombinant, In Vitro, SDS Page, Activation Assay, Purification, Mutagenesis

Comparison of apo APC/C in unphosphorylated and phosphorylated states. a , b , Superposition of the apo unphosphorylated (magenta) and phosphorylated (cyan) APC/C EM maps revealed little conformational differences except in the vicinity of the C-box binding site. c , Apc3A is in an equilibrium between open (light blue) and closed (orange) conformations. While in the inactive apo state, the majority of Apc3A is in the closed state, association of Cdc20 IR stabilizes the open state. d , Sequence alignment of the Apc1 300s loop across different species human, mouse, Xenopus tropicalis (Western clawed frog) and Danio rerio (zebrafish). Phosphorylation sites are indicated and residues 361-380 accounting for the Apc1 AI segment are boxed.
Figure Legend Snippet: Comparison of apo APC/C in unphosphorylated and phosphorylated states. a , b , Superposition of the apo unphosphorylated (magenta) and phosphorylated (cyan) APC/C EM maps revealed little conformational differences except in the vicinity of the C-box binding site. c , Apc3A is in an equilibrium between open (light blue) and closed (orange) conformations. While in the inactive apo state, the majority of Apc3A is in the closed state, association of Cdc20 IR stabilizes the open state. d , Sequence alignment of the Apc1 300s loop across different species human, mouse, Xenopus tropicalis (Western clawed frog) and Danio rerio (zebrafish). Phosphorylation sites are indicated and residues 361-380 accounting for the Apc1 AI segment are boxed.

Techniques Used: Binding Assay, Sequencing, Western Blot

Analytical gel filtration and activity assays. a , With equal amount of input Cdc20, phosphorylated APC/C could form a stable binary complex with Cdc20 after a gel filtration purification step (lane 5), whereas unphosphorylated APC/C could not (lane 4). b , Both unphosphorylated and phosphorylated APC/C associate with Cdh1 stably on gel filtration, as well as APC/C ΔApc1-300s ) served as a loading control and unphosphorylated APC/C alone is used as a negative control for Western-blotting. c , Point mutations of peptide 7 (residues 361-380), either when Arg368 was mutated to glutamate or when the four neighbouring serines were mutated to phospho-mimics (Glu), caused the peptide to abolish its inhibition effect and restored the APC/C activity (lanes 4, 5). Phosphorylation of a single Ser377 only resulted in partial activation of the APC/C (lane 6). d , Chimeric proteins composed of the NTD, the WD40 domain and the IR tail of either Cdc20 or Cdh1 were purified to study their differences in APC/C activation. Both the NTD and the CTD of the coactivators are essential for their association with the APC/C. Swapping both NTD and CTD of Cdh1 with Cdc20 makes it phosphorylation sensitive (lanes 7, 8), similar to Cdc20 (lanes 9, 10) and vice versa . e , Upper panel: Cdh1 can activate both unphosphorylated and phosphorylated APC/C similarly, whereas Cdc20 requires APC/C phosphorylation for its activity. Lower panel: A titration of Cdh1 against unphosphorylated APC/C and APC/C ΔApc1-300s showed enhanced activity in the absence of the Apc1 AI segment at low Cdh1 concentration (≤ 10 nM), whereas Cdc20 requires displacement of the AI segment for its activity. f , Deletion of the Cdh1 α3-helix resulted in reduced activation of the APC/C and makes Cdh1 more phosphorylation sensitive. The substrate Cdk2-cyclin A2-Cks2 was used for assay in c and Hsl1 for the assays in d-f . 20 nM Cdc20 was used in c , 10 nM chimeric coactivators in d and 30 nM coactivators in f . Experiments in a and b were replicated two times, in c, e and f three times and in d for gel source data.
Figure Legend Snippet: Analytical gel filtration and activity assays. a , With equal amount of input Cdc20, phosphorylated APC/C could form a stable binary complex with Cdc20 after a gel filtration purification step (lane 5), whereas unphosphorylated APC/C could not (lane 4). b , Both unphosphorylated and phosphorylated APC/C associate with Cdh1 stably on gel filtration, as well as APC/C ΔApc1-300s ) served as a loading control and unphosphorylated APC/C alone is used as a negative control for Western-blotting. c , Point mutations of peptide 7 (residues 361-380), either when Arg368 was mutated to glutamate or when the four neighbouring serines were mutated to phospho-mimics (Glu), caused the peptide to abolish its inhibition effect and restored the APC/C activity (lanes 4, 5). Phosphorylation of a single Ser377 only resulted in partial activation of the APC/C (lane 6). d , Chimeric proteins composed of the NTD, the WD40 domain and the IR tail of either Cdc20 or Cdh1 were purified to study their differences in APC/C activation. Both the NTD and the CTD of the coactivators are essential for their association with the APC/C. Swapping both NTD and CTD of Cdh1 with Cdc20 makes it phosphorylation sensitive (lanes 7, 8), similar to Cdc20 (lanes 9, 10) and vice versa . e , Upper panel: Cdh1 can activate both unphosphorylated and phosphorylated APC/C similarly, whereas Cdc20 requires APC/C phosphorylation for its activity. Lower panel: A titration of Cdh1 against unphosphorylated APC/C and APC/C ΔApc1-300s showed enhanced activity in the absence of the Apc1 AI segment at low Cdh1 concentration (≤ 10 nM), whereas Cdc20 requires displacement of the AI segment for its activity. f , Deletion of the Cdh1 α3-helix resulted in reduced activation of the APC/C and makes Cdh1 more phosphorylation sensitive. The substrate Cdk2-cyclin A2-Cks2 was used for assay in c and Hsl1 for the assays in d-f . 20 nM Cdc20 was used in c , 10 nM chimeric coactivators in d and 30 nM coactivators in f . Experiments in a and b were replicated two times, in c, e and f three times and in d for gel source data.

Techniques Used: Filtration, Activity Assay, Purification, Stable Transfection, Negative Control, Western Blot, Inhibition, Activation Assay, Titration, Concentration Assay

EM reconstructions of the APC/C Cdc20-Hsl1 complex and comparison of Cdc20 NTD and Cdh1 NTD . a, b , Two views of APC/C Cdc20-Hsl1 shown in cartoon with the D box and Cdc20 IR highlighted in surface representation. Cdc20 binds to the APC/C in juxtaposition to Apc10 to form the substrate recognition module. Apc11 is modelled based on the APC/C Cdh1-Emi1 . c , Both Cdc20 NTD (purple) and Cdh1 NTD (grey, aligned to APC/C Cdc20-Hsl1 interact with Apc1 and Apc8B , whereas Cdh1 NTD contains an additional α3-helix associating with Apc1. I) The crucial C box motif is well conserved between the two coactivators and forms extensive interactions with Apc8B. II) The KLLR motif of Cdh1 is present in the α3-helix to engage Apc1, whereas the related Cdc20 KILR motif contacts Apc8B to augment C-box binding.
Figure Legend Snippet: EM reconstructions of the APC/C Cdc20-Hsl1 complex and comparison of Cdc20 NTD and Cdh1 NTD . a, b , Two views of APC/C Cdc20-Hsl1 shown in cartoon with the D box and Cdc20 IR highlighted in surface representation. Cdc20 binds to the APC/C in juxtaposition to Apc10 to form the substrate recognition module. Apc11 is modelled based on the APC/C Cdh1-Emi1 . c , Both Cdc20 NTD (purple) and Cdh1 NTD (grey, aligned to APC/C Cdc20-Hsl1 interact with Apc1 and Apc8B , whereas Cdh1 NTD contains an additional α3-helix associating with Apc1. I) The crucial C box motif is well conserved between the two coactivators and forms extensive interactions with Apc8B. II) The KLLR motif of Cdh1 is present in the α3-helix to engage Apc1, whereas the related Cdc20 KILR motif contacts Apc8B to augment C-box binding.

Techniques Used: Cytotoxicity Assay, Binding Assay

9) Product Images from "pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA"

Article Title: pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA

Journal: eLife

doi: 10.7554/eLife.08939

Electrophoresis mobility shift assay for influenza virus replication factor-2 (IREF-2) and viral RNA. Radioactively labeled 53-nt-long model vRNA and complementary RNA(cRNA) probes (v53 and c53; 246.9 cpm/fmol) were synthesized by T7 RNA polymerase using [α- 32 P] GTP and isolated by gel excision. Each 500 pM (final concentration) of the labeled viral RNA probes, v53 (lanes 1–7) and c53 (lanes 8–14) was incubated with 10 nM or 50 nM of recombinant NP prepared using the Escherichia. coli expression system (lanes 2, 3, 9, and 10), recombinant pp32 (lanes 4, 5, 11, and 12), and recombinant APRIL (lanes 6, 7, 13, and 14) in 50 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 0.5 U/μl of RNase inhibitor, and 15% (v/v) glycerol at 30°C for 30 min. After incubation, each binding mixture was loaded onto 0.6% agarose gel (buffered with TBE) and separated by electrophoresis (50 V for 3 hr). The gel was dried and visualized by autoradiography. DOI: http://dx.doi.org/10.7554/eLife.08939.008
Figure Legend Snippet: Electrophoresis mobility shift assay for influenza virus replication factor-2 (IREF-2) and viral RNA. Radioactively labeled 53-nt-long model vRNA and complementary RNA(cRNA) probes (v53 and c53; 246.9 cpm/fmol) were synthesized by T7 RNA polymerase using [α- 32 P] GTP and isolated by gel excision. Each 500 pM (final concentration) of the labeled viral RNA probes, v53 (lanes 1–7) and c53 (lanes 8–14) was incubated with 10 nM or 50 nM of recombinant NP prepared using the Escherichia. coli expression system (lanes 2, 3, 9, and 10), recombinant pp32 (lanes 4, 5, 11, and 12), and recombinant APRIL (lanes 6, 7, 13, and 14) in 50 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 0.5 U/μl of RNase inhibitor, and 15% (v/v) glycerol at 30°C for 30 min. After incubation, each binding mixture was loaded onto 0.6% agarose gel (buffered with TBE) and separated by electrophoresis (50 V for 3 hr). The gel was dried and visualized by autoradiography. DOI: http://dx.doi.org/10.7554/eLife.08939.008

Techniques Used: Electrophoresis, Mobility Shift, Labeling, Synthesized, Isolation, Concentration Assay, Incubation, Recombinant, Expressing, Binding Assay, Agarose Gel Electrophoresis, Autoradiography

10) Product Images from "Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication"

Article Title: Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky460

Cleavage of mismatch-containing DNA by Cgl EndoMS-WT and Cgl EndoMS-ΔCter with Cgl β-clamp. ( A ) The 5′-Cy5-labeled DNA substrates (5 nM) containing the GT base pair were incubated with the proteins. The products were analyzed by native 10% PAGE followed by laser scanning (cropped gel image). Lanes 1, positive control (20 nM TkoEndoMS, as a monomer); 2, negative control (no proteins); 3–7, 50 nM Cgl EndoMS-WT (as a monomer); 8–12, 50 nM Cgl EndoMS-ΔCter (as a monomer); 13, no EndoMS. The indicated concentrations (as a monomer) of Cgl β-clamp were added to the reactions. Representative results are shown. The band assignments are indicated on the side of the panels: s, substrates: p, cleaved products. ( B ) Quantification of the cleaved products. Independent data points from three measurements are plotted. ( C ) The physical interactions of Cgl EndoMS with Cgl β-clamp were characterized by SPR using a BIACORE J system (Biacore Inc.). Purified Cgl β-Clamp was immobilized on the sensor Chip CM5, and various concentrations (indicated on the right sides of the sensorgrams) of Cgl EndoMS-WT and Cgl EndoMS-ΔCter were loaded onto the chip for 2 min at a flow rate of 30 μl/min in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Regeneration was achieved using a 1-min injection of 0.025% SDS. Each background response was subtracted. Data were analyzed using BIAevaluation v.3 software (Biacore Inc.)
Figure Legend Snippet: Cleavage of mismatch-containing DNA by Cgl EndoMS-WT and Cgl EndoMS-ΔCter with Cgl β-clamp. ( A ) The 5′-Cy5-labeled DNA substrates (5 nM) containing the GT base pair were incubated with the proteins. The products were analyzed by native 10% PAGE followed by laser scanning (cropped gel image). Lanes 1, positive control (20 nM TkoEndoMS, as a monomer); 2, negative control (no proteins); 3–7, 50 nM Cgl EndoMS-WT (as a monomer); 8–12, 50 nM Cgl EndoMS-ΔCter (as a monomer); 13, no EndoMS. The indicated concentrations (as a monomer) of Cgl β-clamp were added to the reactions. Representative results are shown. The band assignments are indicated on the side of the panels: s, substrates: p, cleaved products. ( B ) Quantification of the cleaved products. Independent data points from three measurements are plotted. ( C ) The physical interactions of Cgl EndoMS with Cgl β-clamp were characterized by SPR using a BIACORE J system (Biacore Inc.). Purified Cgl β-Clamp was immobilized on the sensor Chip CM5, and various concentrations (indicated on the right sides of the sensorgrams) of Cgl EndoMS-WT and Cgl EndoMS-ΔCter were loaded onto the chip for 2 min at a flow rate of 30 μl/min in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Regeneration was achieved using a 1-min injection of 0.025% SDS. Each background response was subtracted. Data were analyzed using BIAevaluation v.3 software (Biacore Inc.)

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Positive Control, Negative Control, SPR Assay, Purification, Chromatin Immunoprecipitation, Flow Cytometry, Injection, Software

11) Product Images from "Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity"

Article Title: Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0166935

Characterization of Siglec-9-EC with the fluorescence-based thermal shift assay. (A) The effect of different buffer pH values on the stability of Siglec-9-EC. The tested buffers were: Citric_pH4 = 100 mM citrate buffer pH 4.0, NaAcetate_pH5 = 100 mM Sodium acetate buffer pH 5.0, MES_pH6 = 100 mM MES buffer pH 6.0, HEPES_pH7 = 100 mM HEPES buffer pH 7.0, Tris-HCl_pH8 = 100 mM Tris-HCl buffer pH 8.0, ImidMaleic_pH 8.5 = 100 mM N-imidazolyl maleamic acid pH 8.5, CHES_pH 9.5 = 100 mM CHES buffer pH 9.5. All of them contained 125 mM NaCl. (B) Thermal shift assay of Siglec-9-EC in 20 mM HEPES buffer, 150mM NaCl, pH 7.4 in the presence of different additives. (C) Thermal shift assay of Siglec-9-EC and the mutant Siglec-9-EC proteins to check the effect of the mutations on the stability of the protein.
Figure Legend Snippet: Characterization of Siglec-9-EC with the fluorescence-based thermal shift assay. (A) The effect of different buffer pH values on the stability of Siglec-9-EC. The tested buffers were: Citric_pH4 = 100 mM citrate buffer pH 4.0, NaAcetate_pH5 = 100 mM Sodium acetate buffer pH 5.0, MES_pH6 = 100 mM MES buffer pH 6.0, HEPES_pH7 = 100 mM HEPES buffer pH 7.0, Tris-HCl_pH8 = 100 mM Tris-HCl buffer pH 8.0, ImidMaleic_pH 8.5 = 100 mM N-imidazolyl maleamic acid pH 8.5, CHES_pH 9.5 = 100 mM CHES buffer pH 9.5. All of them contained 125 mM NaCl. (B) Thermal shift assay of Siglec-9-EC in 20 mM HEPES buffer, 150mM NaCl, pH 7.4 in the presence of different additives. (C) Thermal shift assay of Siglec-9-EC and the mutant Siglec-9-EC proteins to check the effect of the mutations on the stability of the protein.

Techniques Used: Fluorescence, Thermal Shift Assay, Mutagenesis

12) Product Images from "Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication"

Article Title: Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky460

Cleavage of mismatch-containing DNA by Cgl EndoMS-WT and Cgl EndoMS-ΔCter with Cgl β-clamp. ( A ) The 5′-Cy5-labeled DNA substrates (5 nM) containing the GT base pair were incubated with the proteins. The products were analyzed by native 10% PAGE followed by laser scanning (cropped gel image). Lanes 1, positive control (20 nM TkoEndoMS, as a monomer); 2, negative control (no proteins); 3–7, 50 nM Cgl EndoMS-WT (as a monomer); 8–12, 50 nM Cgl EndoMS-ΔCter (as a monomer); 13, no EndoMS. The indicated concentrations (as a monomer) of Cgl β-clamp were added to the reactions. Representative results are shown. The band assignments are indicated on the side of the panels: s, substrates: p, cleaved products. ( B ) Quantification of the cleaved products. Independent data points from three measurements are plotted. ( C ) The physical interactions of Cgl EndoMS with Cgl β-clamp were characterized by SPR using a BIACORE J system (Biacore Inc.). Purified Cgl β-Clamp was immobilized on the sensor Chip CM5, and various concentrations (indicated on the right sides of the sensorgrams) of Cgl EndoMS-WT and Cgl EndoMS-ΔCter were loaded onto the chip for 2 min at a flow rate of 30 μl/min in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Regeneration was achieved using a 1-min injection of 0.025% SDS. Each background response was subtracted. Data were analyzed using BIAevaluation v.3 software (Biacore Inc.)
Figure Legend Snippet: Cleavage of mismatch-containing DNA by Cgl EndoMS-WT and Cgl EndoMS-ΔCter with Cgl β-clamp. ( A ) The 5′-Cy5-labeled DNA substrates (5 nM) containing the GT base pair were incubated with the proteins. The products were analyzed by native 10% PAGE followed by laser scanning (cropped gel image). Lanes 1, positive control (20 nM TkoEndoMS, as a monomer); 2, negative control (no proteins); 3–7, 50 nM Cgl EndoMS-WT (as a monomer); 8–12, 50 nM Cgl EndoMS-ΔCter (as a monomer); 13, no EndoMS. The indicated concentrations (as a monomer) of Cgl β-clamp were added to the reactions. Representative results are shown. The band assignments are indicated on the side of the panels: s, substrates: p, cleaved products. ( B ) Quantification of the cleaved products. Independent data points from three measurements are plotted. ( C ) The physical interactions of Cgl EndoMS with Cgl β-clamp were characterized by SPR using a BIACORE J system (Biacore Inc.). Purified Cgl β-Clamp was immobilized on the sensor Chip CM5, and various concentrations (indicated on the right sides of the sensorgrams) of Cgl EndoMS-WT and Cgl EndoMS-ΔCter were loaded onto the chip for 2 min at a flow rate of 30 μl/min in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Regeneration was achieved using a 1-min injection of 0.025% SDS. Each background response was subtracted. Data were analyzed using BIAevaluation v.3 software (Biacore Inc.)

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Positive Control, Negative Control, SPR Assay, Purification, Chromatin Immunoprecipitation, Flow Cytometry, Injection, Software

13) Product Images from "Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura"

Article Title: Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura

Journal: PLoS ONE

doi: 10.1371/journal.pone.0160388

Characterization of the ex vivo inhibitory effect of anti-mMDTCS mAbs 13B4 and 14H7. Adamts13 +/+ mice (n = 4, per condition) were injected with 2.50 mg/kg of mAb 13B4, 14H7 or 20A10 or with a combination of mAbs 13B4 and 14H7 (1.25 mg/kg each) on day 0 (black arrow). The optimal injection dose of mAb was determined in separate experiments (data not shown). Blood was retrieved 7 days before (‘day -7’) and 1, 3, 5, 7 and 14 days post injection. (A) The influence of the different mAbs on the proteolytic activity of mADAMTS13 was determined using the FRETS-VWF73 assay. Activities were calculated based on the slope of the proteolysis reactions ( S1 Fig ). (B) Plasma mAb levels (μg/mL) were determined using ELISA. Plates were coated with recombinant mADAMST13, blocked and plasma of the respective mice was added. Bound mAbs were detected using GAM-HRP. (C) The amount of mADAMTS13 (%) in plasma was determined using ELISA. Plasma mADAMTS13 was captured using the anti-mT2-CUB2 mAb 9F2. After blocking, the respective plasma samples were added. Finally, bound mADAMTS13 was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (D) Platelet counts were measured of the respective mice samples. Error bars represent the SD (n = 4, per condition). (E) The plasma mVWF multimer pattern was determined for a new cohort of treated mice (n = 5, per condition) 7 days before (‘day -7’) and 1 and 3 days post injection of mAb(s) 20A10 or the combination of mAbs 13B4 and 14H7). Representative multimer patterns are given. Low, middle and high molecular weight (respectively LMW [1–5 bands], MMW [6–10 bands] and HMW [ > 10 bands]) multimers and UL-VWF multimers (brace) are indicated. (F) The percentage HMW multimers was calculated using the ImageJ 1.48v software.
Figure Legend Snippet: Characterization of the ex vivo inhibitory effect of anti-mMDTCS mAbs 13B4 and 14H7. Adamts13 +/+ mice (n = 4, per condition) were injected with 2.50 mg/kg of mAb 13B4, 14H7 or 20A10 or with a combination of mAbs 13B4 and 14H7 (1.25 mg/kg each) on day 0 (black arrow). The optimal injection dose of mAb was determined in separate experiments (data not shown). Blood was retrieved 7 days before (‘day -7’) and 1, 3, 5, 7 and 14 days post injection. (A) The influence of the different mAbs on the proteolytic activity of mADAMTS13 was determined using the FRETS-VWF73 assay. Activities were calculated based on the slope of the proteolysis reactions ( S1 Fig ). (B) Plasma mAb levels (μg/mL) were determined using ELISA. Plates were coated with recombinant mADAMST13, blocked and plasma of the respective mice was added. Bound mAbs were detected using GAM-HRP. (C) The amount of mADAMTS13 (%) in plasma was determined using ELISA. Plasma mADAMTS13 was captured using the anti-mT2-CUB2 mAb 9F2. After blocking, the respective plasma samples were added. Finally, bound mADAMTS13 was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (D) Platelet counts were measured of the respective mice samples. Error bars represent the SD (n = 4, per condition). (E) The plasma mVWF multimer pattern was determined for a new cohort of treated mice (n = 5, per condition) 7 days before (‘day -7’) and 1 and 3 days post injection of mAb(s) 20A10 or the combination of mAbs 13B4 and 14H7). Representative multimer patterns are given. Low, middle and high molecular weight (respectively LMW [1–5 bands], MMW [6–10 bands] and HMW [ > 10 bands]) multimers and UL-VWF multimers (brace) are indicated. (F) The percentage HMW multimers was calculated using the ImageJ 1.48v software.

Techniques Used: Ex Vivo, Mouse Assay, Injection, Activity Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Molecular Weight, Software

Development of a sensitive mADAMTS13 detection assay. (A) Binding of plasma mADAMTS13 to the anti-mADAMTS13 mAbs was tested in ELISA. The respective anti-mMDTCS (black) and anti-mT2-CUB2 (white) mAbs were coated on a 96-well microtiter plate. After blocking, plasma mADAMTS13 was added (0.1 U/mL) and was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (B) The same ELISA was performed as in A in triplicate for anti-mT2-CUB2 mAb 9F2, anti-mMDTCS mAb 14H7 and the previously reported anti-mMDTCS mAb 20A10 [ 31 ]. Binding was calculated relative to binding of 0.10 U/mL plasma mADAMTS13 to 20A10 (which was set to a value of ‘1.0’). Error bars represent the SD of the three independently performed experiments.
Figure Legend Snippet: Development of a sensitive mADAMTS13 detection assay. (A) Binding of plasma mADAMTS13 to the anti-mADAMTS13 mAbs was tested in ELISA. The respective anti-mMDTCS (black) and anti-mT2-CUB2 (white) mAbs were coated on a 96-well microtiter plate. After blocking, plasma mADAMTS13 was added (0.1 U/mL) and was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (B) The same ELISA was performed as in A in triplicate for anti-mT2-CUB2 mAb 9F2, anti-mMDTCS mAb 14H7 and the previously reported anti-mMDTCS mAb 20A10 [ 31 ]. Binding was calculated relative to binding of 0.10 U/mL plasma mADAMTS13 to 20A10 (which was set to a value of ‘1.0’). Error bars represent the SD of the three independently performed experiments.

Techniques Used: Detection Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

A subset of anti-mMDTCS mAbs inhibit plasma mADAMTS13 activity. The effect of anti-mADAMTS13 mAbs on the proteolytic activity of plasma mADAMTS13 was tested in vitro using the FRETS-VWF73 assay. (A) All anti-mADAMTS13 mAbs, both anti-mMDTCS and anti-mT2-CUB2 mAbs, were tested. For each condition, the activity of plasma mADAMTS13 was calculated using a calibration curve of 2.5%, 5%, 10% and 15% NMP. (B) Time profile of the cleavage of the FRETS-VWF73 substrate by plasma mADAMTS13, in the absence or presence of mAbs 13B4 and/or 14H7. Error bars represent the SD of at least three independently performed experiments.
Figure Legend Snippet: A subset of anti-mMDTCS mAbs inhibit plasma mADAMTS13 activity. The effect of anti-mADAMTS13 mAbs on the proteolytic activity of plasma mADAMTS13 was tested in vitro using the FRETS-VWF73 assay. (A) All anti-mADAMTS13 mAbs, both anti-mMDTCS and anti-mT2-CUB2 mAbs, were tested. For each condition, the activity of plasma mADAMTS13 was calculated using a calibration curve of 2.5%, 5%, 10% and 15% NMP. (B) Time profile of the cleavage of the FRETS-VWF73 substrate by plasma mADAMTS13, in the absence or presence of mAbs 13B4 and/or 14H7. Error bars represent the SD of at least three independently performed experiments.

Techniques Used: Activity Assay, In Vitro

Epitope mapping and epitope overview of the developed anti-mADAMTS13 mAbs. The epitope of each anti-mADAMTS13 mAb was mapped against both mMDTCS (A) and mT2-CUB2 (B). Individual anti-mADAMTS13 mAbs were coated, recombinant mMDTCS (A) or mT2-CUB2 (B) were added and binding of the respective mADAMTS13 variant was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. Black and white bars represent respectively anti-mMDTCS and anti-mT2-CUB2 mAbs. The previously reported mAb 20A10 [ 31 ] was used as a positive (A) and negative (B) control. (C) Epitope overview of the developed anti-mADAMTS13 mAbs. The previously developed mAb 20A10 [ 31 ] is marked by a dark frame.
Figure Legend Snippet: Epitope mapping and epitope overview of the developed anti-mADAMTS13 mAbs. The epitope of each anti-mADAMTS13 mAb was mapped against both mMDTCS (A) and mT2-CUB2 (B). Individual anti-mADAMTS13 mAbs were coated, recombinant mMDTCS (A) or mT2-CUB2 (B) were added and binding of the respective mADAMTS13 variant was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. Black and white bars represent respectively anti-mMDTCS and anti-mT2-CUB2 mAbs. The previously reported mAb 20A10 [ 31 ] was used as a positive (A) and negative (B) control. (C) Epitope overview of the developed anti-mADAMTS13 mAbs. The previously developed mAb 20A10 [ 31 ] is marked by a dark frame.

Techniques Used: Recombinant, Binding Assay, Variant Assay

14) Product Images from "Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP"

Article Title: Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP

Journal: Acta Crystallographica. Section F, Structural Biology Communications

doi: 10.1107/S2053230X13034705

Melting curve. A temperature melting curve of AtzF was performed in triplicate, using differential scanning fluorimetry with the dye SYPRO Orange. The red curves are the AtzF protein (in triplicate) in the buffer used for crystallization (50 m M HEPES, 100 m M NaCl pH 7.5). The blue curves are a 0.1 mg ml −1 lysozyme control.
Figure Legend Snippet: Melting curve. A temperature melting curve of AtzF was performed in triplicate, using differential scanning fluorimetry with the dye SYPRO Orange. The red curves are the AtzF protein (in triplicate) in the buffer used for crystallization (50 m M HEPES, 100 m M NaCl pH 7.5). The blue curves are a 0.1 mg ml −1 lysozyme control.

Techniques Used: Crystallization Assay

15) Product Images from "Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module"

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

Journal: Nature cell biology

doi: 10.1038/ncb3241

Regulation of TPX2 and chTOG-stimulated microtubule nucleation by importins (a) Time series of TIRF microscopy images showing microtubule nucleation and growth on surfaces with immobilised biotinylated rigor kinesin (pre-incubated at 125 nM) always in the presence of 100 nM chTOG and Atto647N-labelled tubulin, without and with additional 500 nM importin α/ β complex (first and second row, respectively), 100 nM mGFP-TPX2 (third row) or both 500 nM importin α/β and 100 nM mGFP-TPX2 (fourth row) as indicated. Plots showing the time course of the mean Atto647N-labelled microtubule (magenta) and mGFP-TPX2 (green) intensities for the same experiments are shown next to each individual times series of images. Note that the mGFP-TPX2 signal declines at later time points likely due to depletion of TPX2 from solution due to binding to the efficiently nucleated growing microtubules. (b) Time series of TIRF microscopy images showing that compared to a control (left), inclusion of importin α/β (500 nM) inhibits stabilisation of Atto647N-labelled nucleation intermediates by surface-immobilised biotinylated TPX2 (pre-incubated at 125 nM) (right image pair). Plot showing the time course of the mean Atto647N-labelled tubulin intensities on the surface (right). Atto647N-labelled tubulin concentration was always 12.5 μM. Scale bars as indicated. t = 0 when the sample is placed at 30°C. (c) Model of synergistic TPX2 and chTOG-stimulated microtubule nucleation and growth. Different reactions, all promoting nucleation and growth, are regulated by the distinct and complementary activities of TPX2 and chTOG.
Figure Legend Snippet: Regulation of TPX2 and chTOG-stimulated microtubule nucleation by importins (a) Time series of TIRF microscopy images showing microtubule nucleation and growth on surfaces with immobilised biotinylated rigor kinesin (pre-incubated at 125 nM) always in the presence of 100 nM chTOG and Atto647N-labelled tubulin, without and with additional 500 nM importin α/ β complex (first and second row, respectively), 100 nM mGFP-TPX2 (third row) or both 500 nM importin α/β and 100 nM mGFP-TPX2 (fourth row) as indicated. Plots showing the time course of the mean Atto647N-labelled microtubule (magenta) and mGFP-TPX2 (green) intensities for the same experiments are shown next to each individual times series of images. Note that the mGFP-TPX2 signal declines at later time points likely due to depletion of TPX2 from solution due to binding to the efficiently nucleated growing microtubules. (b) Time series of TIRF microscopy images showing that compared to a control (left), inclusion of importin α/β (500 nM) inhibits stabilisation of Atto647N-labelled nucleation intermediates by surface-immobilised biotinylated TPX2 (pre-incubated at 125 nM) (right image pair). Plot showing the time course of the mean Atto647N-labelled tubulin intensities on the surface (right). Atto647N-labelled tubulin concentration was always 12.5 μM. Scale bars as indicated. t = 0 when the sample is placed at 30°C. (c) Model of synergistic TPX2 and chTOG-stimulated microtubule nucleation and growth. Different reactions, all promoting nucleation and growth, are regulated by the distinct and complementary activities of TPX2 and chTOG.

Techniques Used: Microscopy, Incubation, Binding Assay, Concentration Assay

The central part of human TPX2 determines its binding preference for growing microtubule ends (a) Scheme of the human TPX2 constructs used in this study: full-length TPX2, N-terminally truncated TPX2 containing amino acids 274 - 747 (TPX2 ΔN ), and a minimal TPX2 construct containing amino acids 274 - 659 (TPX2 mini ). Regions known to interact with Aurora A (AurA), importin α (Imp α) are indicated, together with predicted coil coils (CC) and nuclear localisation signal (NLS). (b) Coomassie Blue-stained SDS-PAGE gel showing 1 μg of purified recombinant TPX2 constructs. (c, d) Single channel and merged TIRF microscopy images showing how mGFP-TPX2 (green in merge) at 5 nM (c) and 0.35 nM (d) binds either all along (c) or preferentially to the growing ends and the GMPCPP segment (d) of a growing Atto647N-labelled microtubule (magenta in merge) (“−“ and “+” indicate end binding, the GMPCPP “seed” is marked by an arrowhead). (e) Kymographs depicting the time course of binding of 5 nM mGFP-TPX2 all along a growing microtubule. Att647N-labelled tubulin concentration was 7.5 μM. (f) Kymographs depicting the time course of 0.35 nM mGFP-TPX2 binding to a growing microtubule end and the GMPCPP “seed”. Att647N-labelled tubulin concentration was 12.5 μM. (g, h) Kymographs showing binding of (g) 10 nM mGFP-TPX2 ΔN and (h) 33 nM mGFP-TPX2 mini to dynamic microtubules (merged channels on the left, mGFP-TPX2 on the right). Atto647N-labelled tubulin concentration is 12.5 and 15 μM, respectively. (i, j) Kymographs showing that neither (i) 1 nM full-length mGFP-TPX2 nor (j) 10 nM TPX2 mini binds to shrinking microtubules. Atto647N-labelled tubulin concentrations were 5 μM and 7.5 μM, respectively. For all kymograph pairs: merged channel - left, mGFP-TPX2 channel - right. Scale bars as indicated.
Figure Legend Snippet: The central part of human TPX2 determines its binding preference for growing microtubule ends (a) Scheme of the human TPX2 constructs used in this study: full-length TPX2, N-terminally truncated TPX2 containing amino acids 274 - 747 (TPX2 ΔN ), and a minimal TPX2 construct containing amino acids 274 - 659 (TPX2 mini ). Regions known to interact with Aurora A (AurA), importin α (Imp α) are indicated, together with predicted coil coils (CC) and nuclear localisation signal (NLS). (b) Coomassie Blue-stained SDS-PAGE gel showing 1 μg of purified recombinant TPX2 constructs. (c, d) Single channel and merged TIRF microscopy images showing how mGFP-TPX2 (green in merge) at 5 nM (c) and 0.35 nM (d) binds either all along (c) or preferentially to the growing ends and the GMPCPP segment (d) of a growing Atto647N-labelled microtubule (magenta in merge) (“−“ and “+” indicate end binding, the GMPCPP “seed” is marked by an arrowhead). (e) Kymographs depicting the time course of binding of 5 nM mGFP-TPX2 all along a growing microtubule. Att647N-labelled tubulin concentration was 7.5 μM. (f) Kymographs depicting the time course of 0.35 nM mGFP-TPX2 binding to a growing microtubule end and the GMPCPP “seed”. Att647N-labelled tubulin concentration was 12.5 μM. (g, h) Kymographs showing binding of (g) 10 nM mGFP-TPX2 ΔN and (h) 33 nM mGFP-TPX2 mini to dynamic microtubules (merged channels on the left, mGFP-TPX2 on the right). Atto647N-labelled tubulin concentration is 12.5 and 15 μM, respectively. (i, j) Kymographs showing that neither (i) 1 nM full-length mGFP-TPX2 nor (j) 10 nM TPX2 mini binds to shrinking microtubules. Atto647N-labelled tubulin concentrations were 5 μM and 7.5 μM, respectively. For all kymograph pairs: merged channel - left, mGFP-TPX2 channel - right. Scale bars as indicated.

Techniques Used: Binding Assay, Construct, Staining, SDS Page, Purification, Recombinant, Microscopy, Concentration Assay

16) Product Images from "The Saccharomyces cerevisiae Telomerase Subunit Est3 Binds Telomeres in a Cell Cycle- and Est1-Dependent Manner and Interacts Directly with Est1 In Vitro"

Article Title: The Saccharomyces cerevisiae Telomerase Subunit Est3 Binds Telomeres in a Cell Cycle- and Est1-Dependent Manner and Interacts Directly with Est1 In Vitro

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002060

The late S/G2 phase telomere association of Est1 and Est2 is not reduced in the absence of est3 Δ. Methods are the same as for Figure 1 . (A) Representative FACS analysis from one of three biological replicates from WT and est3 Δ cells expressing Myc-tagged Est1. (B) Quantitative real-time PCR analysis of Myc-tagged Est1 telomere association. The amount of telomere associated Est1 at late S/G2 phase was indistinguishable between WT and est3 Δ cells. (C) Anti-Myc western blot analysis from whole cell protein extracts from asynchronous cultures expressing Myc-tagged Est1. (D) Representative FACS analysis from one of three biological replicates from WT and est3 Δ cells expressing Myc-tagged Est2. (E) Quantitative real-time PCR analysis of Myc-tagged Est2 telomere association. The amount of telomere associated Est2 at late S/G2 phase was indistinguishable between WT and est3 Δ cells throughout the cell cycle at both telomeres except at 30 min (p = 0.05) for TEL VI-R. (F) Anti-Myc western blot analysis of whole cell protein extracts from asynchronous cells expressing Myc-tagged Est2.
Figure Legend Snippet: The late S/G2 phase telomere association of Est1 and Est2 is not reduced in the absence of est3 Δ. Methods are the same as for Figure 1 . (A) Representative FACS analysis from one of three biological replicates from WT and est3 Δ cells expressing Myc-tagged Est1. (B) Quantitative real-time PCR analysis of Myc-tagged Est1 telomere association. The amount of telomere associated Est1 at late S/G2 phase was indistinguishable between WT and est3 Δ cells. (C) Anti-Myc western blot analysis from whole cell protein extracts from asynchronous cultures expressing Myc-tagged Est1. (D) Representative FACS analysis from one of three biological replicates from WT and est3 Δ cells expressing Myc-tagged Est2. (E) Quantitative real-time PCR analysis of Myc-tagged Est2 telomere association. The amount of telomere associated Est2 at late S/G2 phase was indistinguishable between WT and est3 Δ cells throughout the cell cycle at both telomeres except at 30 min (p = 0.05) for TEL VI-R. (F) Anti-Myc western blot analysis of whole cell protein extracts from asynchronous cells expressing Myc-tagged Est2.

Techniques Used: FACS, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Telomere association of Est3 at late S/G2 phase is reduced in the absence of EST1 . Methods are the same as for Figure 1 . (A) Representative FACS analysis from one of four biological replicates from est1 Δ and WT strains expressing Myc-tagged Est3. (B) Quantitative real-time PCR analysis of Myc-tagged Est3 telomere association from untagged, WT and est1 Δ cells. The amount of telomere associated Est3 at G1 phase in est1 Δ cells was low but still significantly higher than in untagged cells at TEL-VI-R (0 min, P = 0.024; 15 min, P = 0.002; 30 min, P = 0.023) and at TEL-XV-L (0 min, P = 0.044; 15 min, P = 0.035). At early S-phase (30 min), Est3 telomere association was significant in est1 Δ cells compared to the untagged strain at TEL-VI-R (P = 0.023) but not at TEL-XV-L (P = 0.098). In est1 Δ cells, the amount of telomere associated Est3 at late S/G2 phase was significantly reduced at TEL VI-R (60 min, P = 0.0007) and at TEL XV-L (60 min, P = 0.0028) compared to WT. The amount of telomere associated Est3 later in the cell cycle was significant compared to the untagged strain at TEL-VI-R (45 min, P = 0.02; 60 min, P = 0.04; 75 min, P = 0.03; 90 min, P = 0.04) but not at TEL-XV-L (45 min, P = 0.09; 60 min, P = 0.07; 75 min, P = 0.46; 90 min, P = 0.58). (C) Anti-Myc western blots from whole cell protein extracts from asynchronous est1 Δ, untagged (unt) and WT (WT) strains expressing Myc-tagged Est3.
Figure Legend Snippet: Telomere association of Est3 at late S/G2 phase is reduced in the absence of EST1 . Methods are the same as for Figure 1 . (A) Representative FACS analysis from one of four biological replicates from est1 Δ and WT strains expressing Myc-tagged Est3. (B) Quantitative real-time PCR analysis of Myc-tagged Est3 telomere association from untagged, WT and est1 Δ cells. The amount of telomere associated Est3 at G1 phase in est1 Δ cells was low but still significantly higher than in untagged cells at TEL-VI-R (0 min, P = 0.024; 15 min, P = 0.002; 30 min, P = 0.023) and at TEL-XV-L (0 min, P = 0.044; 15 min, P = 0.035). At early S-phase (30 min), Est3 telomere association was significant in est1 Δ cells compared to the untagged strain at TEL-VI-R (P = 0.023) but not at TEL-XV-L (P = 0.098). In est1 Δ cells, the amount of telomere associated Est3 at late S/G2 phase was significantly reduced at TEL VI-R (60 min, P = 0.0007) and at TEL XV-L (60 min, P = 0.0028) compared to WT. The amount of telomere associated Est3 later in the cell cycle was significant compared to the untagged strain at TEL-VI-R (45 min, P = 0.02; 60 min, P = 0.04; 75 min, P = 0.03; 90 min, P = 0.04) but not at TEL-XV-L (45 min, P = 0.09; 60 min, P = 0.07; 75 min, P = 0.46; 90 min, P = 0.58). (C) Anti-Myc western blots from whole cell protein extracts from asynchronous est1 Δ, untagged (unt) and WT (WT) strains expressing Myc-tagged Est3.

Techniques Used: FACS, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Est1 and Est3 interact directly in vitro . (A) 1 µg of purified Strep-tagged Est1 (left panel) and tag-removed Est3 (right panel) was resolved on SDS-PAGE gels followed by staining with Coomassie brilliant blue. (B) Interaction between purified Est1 and Est3 was assessed by magnetic beads pull-down assay. The beads fractions is shown in lanes 4-6 (“beads”), and 15% of the input materials is shown as a control in lanes 1–3 (“15% input”). Positions of Est1, BSA, and Est3 are indicated on the right. Proteins were separated on a 10% SDS-PAGE gel and visualized by Coomassie blue staining.
Figure Legend Snippet: Est1 and Est3 interact directly in vitro . (A) 1 µg of purified Strep-tagged Est1 (left panel) and tag-removed Est3 (right panel) was resolved on SDS-PAGE gels followed by staining with Coomassie brilliant blue. (B) Interaction between purified Est1 and Est3 was assessed by magnetic beads pull-down assay. The beads fractions is shown in lanes 4-6 (“beads”), and 15% of the input materials is shown as a control in lanes 1–3 (“15% input”). Positions of Est1, BSA, and Est3 are indicated on the right. Proteins were separated on a 10% SDS-PAGE gel and visualized by Coomassie blue staining.

Techniques Used: In Vitro, Purification, SDS Page, Staining, Magnetic Beads, Pull Down Assay

17) Product Images from "Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A"

Article Title: Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp157

Human mtTFA is an asymmetric monomer in the absence of DNA. ( A ) Deletion constructs of h-mtTFA. The upper panel shows a schematic diagram and the lower panel shows SDS–PAGE of h-mtTFA deletion constructs on a 15% polyacrylamide gel. ( B ) Size-exclusion chromatography (Superdex 200; GE Healthcare) elution profiles of h-mtTFA and h-mtTFA deletion constructs, mtTFA 1–179 , mtTFA 1–109 , mtTFA 1–79 , mtTFA 80–204 , mtTFA 110–204 and mtTFA 110–179 (top panel), and the single HMG domains, HMGB1 box A and HMGD (lower panel) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The position of each size standard is indicated by arrows above the top panel for amylase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and RNase A (14 kDa). The void volume was at 45 ml and is not shown. ( C ) Sedimentation velocity profiles for the raw data acquired at different time points and the residuals after fittings had been performed using SEDFIT in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. ( D ) Calculated sedimentation coefficient distributions for the full-length h-mtTFA.
Figure Legend Snippet: Human mtTFA is an asymmetric monomer in the absence of DNA. ( A ) Deletion constructs of h-mtTFA. The upper panel shows a schematic diagram and the lower panel shows SDS–PAGE of h-mtTFA deletion constructs on a 15% polyacrylamide gel. ( B ) Size-exclusion chromatography (Superdex 200; GE Healthcare) elution profiles of h-mtTFA and h-mtTFA deletion constructs, mtTFA 1–179 , mtTFA 1–109 , mtTFA 1–79 , mtTFA 80–204 , mtTFA 110–204 and mtTFA 110–179 (top panel), and the single HMG domains, HMGB1 box A and HMGD (lower panel) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The position of each size standard is indicated by arrows above the top panel for amylase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and RNase A (14 kDa). The void volume was at 45 ml and is not shown. ( C ) Sedimentation velocity profiles for the raw data acquired at different time points and the residuals after fittings had been performed using SEDFIT in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. ( D ) Calculated sedimentation coefficient distributions for the full-length h-mtTFA.

Techniques Used: Construct, SDS Page, Size-exclusion Chromatography, Sedimentation

Human mtTFA box B interacts with other regions of mtTFA. An N-terminal GST fusion with box B (mtTFA 110–179 ) was tested for its ability to interact with the various deletion constructs of h-mtTFA (mtTFA 1-109 , mtTFA 1–79 , mtTFA 1–80–204 , mtTFA 110–204 , mtTFA 110–179 , mtTFA 80–179 , mtTFA 1–95 and mtTFA 96–179 ) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The reactions were electrophoresed on a 15% SDS–PAGE gel and transferred to nitrocellulose membrane (Millipore) and probed with anti-His and anti-GST antibodies, respectively.
Figure Legend Snippet: Human mtTFA box B interacts with other regions of mtTFA. An N-terminal GST fusion with box B (mtTFA 110–179 ) was tested for its ability to interact with the various deletion constructs of h-mtTFA (mtTFA 1-109 , mtTFA 1–79 , mtTFA 1–80–204 , mtTFA 110–204 , mtTFA 110–179 , mtTFA 80–179 , mtTFA 1–95 and mtTFA 96–179 ) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The reactions were electrophoresed on a 15% SDS–PAGE gel and transferred to nitrocellulose membrane (Millipore) and probed with anti-His and anti-GST antibodies, respectively.

Techniques Used: Construct, SDS Page

18) Product Images from "Intermolecular base stacking mediates RNA-RNA interaction in a crystal structure of the RNA chaperone Hfq"

Article Title: Intermolecular base stacking mediates RNA-RNA interaction in a crystal structure of the RNA chaperone Hfq

Journal: Scientific Reports

doi: 10.1038/s41598-017-10085-8

OxyS and fhlA can interact via their (ARN) X motifs. AUC curves of Hfq102 complexes with the (ARN) X motifs of OxyS and fhlA . All individual species sediment according to their expected sedimentation coefficients and binary complexes sediment as single monomeric Hfq102 6 :RNA species ( a ), but the ternary complex of Hfq102- fhlA -OxyS forms a higher order assembly (black in b ) likely corresponding to Hfq-RNA dimers. This peak is absent with the Oxy0 variant that lacks the N-site bases.
Figure Legend Snippet: OxyS and fhlA can interact via their (ARN) X motifs. AUC curves of Hfq102 complexes with the (ARN) X motifs of OxyS and fhlA . All individual species sediment according to their expected sedimentation coefficients and binary complexes sediment as single monomeric Hfq102 6 :RNA species ( a ), but the ternary complex of Hfq102- fhlA -OxyS forms a higher order assembly (black in b ) likely corresponding to Hfq-RNA dimers. This peak is absent with the Oxy0 variant that lacks the N-site bases.

Techniques Used: Sedimentation, Variant Assay

19) Product Images from "Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products"

Article Title: Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products

Journal: Blood Advances

doi: 10.1182/bloodadvances.2018030452

VWF peptides identified in a MAPPs assay and effect of co-incubation with different FVIII products. (A) For each donor, number of VWF peptides identified in the MAPPs assay is shown by the bars; pdVWF was coincubated with either FL-rFVIII (red) or pdFVIII (blue). Left panel, The MHC-II-DRB1 alleles for each donor and their respective frequencies in African, Asian, and white populations. (B) Location of the VWF peptides identified in the MAPPs assay shown on the VWF primary sequence (x-axis). At each location, the subjects are identified by the color codes.
Figure Legend Snippet: VWF peptides identified in a MAPPs assay and effect of co-incubation with different FVIII products. (A) For each donor, number of VWF peptides identified in the MAPPs assay is shown by the bars; pdVWF was coincubated with either FL-rFVIII (red) or pdFVIII (blue). Left panel, The MHC-II-DRB1 alleles for each donor and their respective frequencies in African, Asian, and white populations. (B) Location of the VWF peptides identified in the MAPPs assay shown on the VWF primary sequence (x-axis). At each location, the subjects are identified by the color codes.

Techniques Used: Incubation, Sequencing

Comparison of FVIII peptides identified in a MAPPs assay following incubation of MoDCs with FL-rFVIII plus pdVWF or pdFVIII plus pdVWF. (A) For each donor and HA patient, number of peptides found in the MAPPs assay for FL-rFVIII plus pdVWF (red) and pdFVIII plus pdVWF (blue) are shown. In addition, the MHC-II–DRB1 alleles for each donor are identified as well as their respective frequencies in African, Asian, and white populations. ( B) Location and frequencies of peptides for a single donor (D1179) identified in the MAPPs assay following incubation of APCs with FL-rFVIII plus pdVWF (blue) or pdFVIII plus pdVWF (red). (C) Pooled data for all subjects showing location and frequencies of peptides identified following incubation with FL-rFVIII plus pdVWF where no peptides were found after incubation with pdFVIII plus pdVWF.
Figure Legend Snippet: Comparison of FVIII peptides identified in a MAPPs assay following incubation of MoDCs with FL-rFVIII plus pdVWF or pdFVIII plus pdVWF. (A) For each donor and HA patient, number of peptides found in the MAPPs assay for FL-rFVIII plus pdVWF (red) and pdFVIII plus pdVWF (blue) are shown. In addition, the MHC-II–DRB1 alleles for each donor are identified as well as their respective frequencies in African, Asian, and white populations. ( B) Location and frequencies of peptides for a single donor (D1179) identified in the MAPPs assay following incubation of APCs with FL-rFVIII plus pdVWF (blue) or pdFVIII plus pdVWF (red). (C) Pooled data for all subjects showing location and frequencies of peptides identified following incubation with FL-rFVIII plus pdVWF where no peptides were found after incubation with pdFVIII plus pdVWF.

Techniques Used: Incubation

Mapping of the MAPPs assay derived peptides on FVIII structure. (pink). (B) Structure depicted in panel A with the regions yielding MHC-II–presented FVIII-derived peptides unique to FL-rFVIII (green). Peptides in green (see domain labels for peptide location) were identified on MoDCs incubated with FL-rFVIII plus pdVWF but not identified when MoDCs from the same subject were incubated with pdFVIII plus pdVWF (ie, protected in the pdFVIII protein). The FVIII domains are identified on the figure in bold letters.
Figure Legend Snippet: Mapping of the MAPPs assay derived peptides on FVIII structure. (pink). (B) Structure depicted in panel A with the regions yielding MHC-II–presented FVIII-derived peptides unique to FL-rFVIII (green). Peptides in green (see domain labels for peptide location) were identified on MoDCs incubated with FL-rFVIII plus pdVWF but not identified when MoDCs from the same subject were incubated with pdFVIII plus pdVWF (ie, protected in the pdFVIII protein). The FVIII domains are identified on the figure in bold letters.

Techniques Used: Derivative Assay, Incubation

20) Product Images from "Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products"

Article Title: Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products

Journal: Blood Advances

doi: 10.1182/bloodadvances.2018030452

VWF peptides identified in a MAPPs assay and effect of co-incubation with different FVIII products. (A) For each donor, number of VWF peptides identified in the MAPPs assay is shown by the bars; pdVWF was coincubated with either FL-rFVIII (red) or pdFVIII (blue). Left panel, The MHC-II-DRB1 alleles for each donor and their respective frequencies in African, Asian, and white populations. (B) Location of the VWF peptides identified in the MAPPs assay shown on the VWF primary sequence (x-axis). At each location, the subjects are identified by the color codes.
Figure Legend Snippet: VWF peptides identified in a MAPPs assay and effect of co-incubation with different FVIII products. (A) For each donor, number of VWF peptides identified in the MAPPs assay is shown by the bars; pdVWF was coincubated with either FL-rFVIII (red) or pdFVIII (blue). Left panel, The MHC-II-DRB1 alleles for each donor and their respective frequencies in African, Asian, and white populations. (B) Location of the VWF peptides identified in the MAPPs assay shown on the VWF primary sequence (x-axis). At each location, the subjects are identified by the color codes.

Techniques Used: Incubation, Sequencing

Comparison of FVIII peptides identified in a MAPPs assay following incubation of MoDCs with FL-rFVIII plus pdVWF or pdFVIII plus pdVWF. (A) For each donor and HA patient, number of peptides found in the MAPPs assay for FL-rFVIII plus pdVWF (red) and pdFVIII plus pdVWF (blue) are shown. In addition, the MHC-II–DRB1 alleles for each donor are identified as well as their respective frequencies in African, Asian, and white populations. ( B) Location and frequencies of peptides for a single donor (D1179) identified in the MAPPs assay following incubation of APCs with FL-rFVIII plus pdVWF (blue) or pdFVIII plus pdVWF (red). (C) Pooled data for all subjects showing location and frequencies of peptides identified following incubation with FL-rFVIII plus pdVWF where no peptides were found after incubation with pdFVIII plus pdVWF.
Figure Legend Snippet: Comparison of FVIII peptides identified in a MAPPs assay following incubation of MoDCs with FL-rFVIII plus pdVWF or pdFVIII plus pdVWF. (A) For each donor and HA patient, number of peptides found in the MAPPs assay for FL-rFVIII plus pdVWF (red) and pdFVIII plus pdVWF (blue) are shown. In addition, the MHC-II–DRB1 alleles for each donor are identified as well as their respective frequencies in African, Asian, and white populations. ( B) Location and frequencies of peptides for a single donor (D1179) identified in the MAPPs assay following incubation of APCs with FL-rFVIII plus pdVWF (blue) or pdFVIII plus pdVWF (red). (C) Pooled data for all subjects showing location and frequencies of peptides identified following incubation with FL-rFVIII plus pdVWF where no peptides were found after incubation with pdFVIII plus pdVWF.

Techniques Used: Incubation

Mapping of the MAPPs assay derived peptides on FVIII structure. (pink). (B) Structure depicted in panel A with the regions yielding MHC-II–presented FVIII-derived peptides unique to FL-rFVIII (green). Peptides in green (see domain labels for peptide location) were identified on MoDCs incubated with FL-rFVIII plus pdVWF but not identified when MoDCs from the same subject were incubated with pdFVIII plus pdVWF (ie, protected in the pdFVIII protein). The FVIII domains are identified on the figure in bold letters.
Figure Legend Snippet: Mapping of the MAPPs assay derived peptides on FVIII structure. (pink). (B) Structure depicted in panel A with the regions yielding MHC-II–presented FVIII-derived peptides unique to FL-rFVIII (green). Peptides in green (see domain labels for peptide location) were identified on MoDCs incubated with FL-rFVIII plus pdVWF but not identified when MoDCs from the same subject were incubated with pdFVIII plus pdVWF (ie, protected in the pdFVIII protein). The FVIII domains are identified on the figure in bold letters.

Techniques Used: Derivative Assay, Incubation

21) Product Images from "Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module"

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

Journal: Nature cell biology

doi: 10.1038/ncb3241

Surface-immobilised TPX2 arrests nucleation intermediates, but in combination with chTOG efficiently nucleates microtubules (a) Scheme of the experimental setup for the ‘surface’ nucleation assay. (b) TIRF microscopy images from a time series showing Atto647N-labelled microtubules and/or tubulin particles on a glass surface with immobilised biotinylated Kin1 rigor mutant, chTOG or TPX2 after pre-incubation at the indicated concentrations. Insets on the right depict magnified images of the same surface at 7.5 min. (c) Time courses of the mean Atto647N-tubulin fluorescence intensities measured at the surface for the entire field of view for different chTOG, TPX2 and Kin1 rigor densities. Concentrations of biotinylated proteins used for surface incubation as indicated. The same Kin1 rigor curve is shown as a control for both chTOG and TPX2 plots. Note that the fluorescence signal typically represents the sum of several different tubulin species: soluble tubulin in the TIRF field (creating part of the background), immobilised individual tubulins (e.g. bound to chTOG), tubulin ‘stubs’ (bound to TPX2) and microtubules (bound to chTOG or TPX2). Raw intensities including background are shown. (d) Time series of TIRF microscopy images showing nucleation and growth of Atto647N-labelled microtubules on surfaces with immobilised biotinylated TPX2 (pre-incubated at 125 nM, top three rows) or, for controls, with a biotinylated Kin1 rigor (pre-incubated at 125 nM, bottom two rows) in either the absence or presence of 100 nM untagged chTOG, as indicated. Because of the high microtubule density at the TPX2 surface in the presence of chTOG, the same time series is also shown with reduced contrast for better visualisation of individual microtubules. Atto647N-labelled tubulin concentration was always 12.5 μM. Scale bars as indicated. t = 0 when the sample is placed at 30°C.
Figure Legend Snippet: Surface-immobilised TPX2 arrests nucleation intermediates, but in combination with chTOG efficiently nucleates microtubules (a) Scheme of the experimental setup for the ‘surface’ nucleation assay. (b) TIRF microscopy images from a time series showing Atto647N-labelled microtubules and/or tubulin particles on a glass surface with immobilised biotinylated Kin1 rigor mutant, chTOG or TPX2 after pre-incubation at the indicated concentrations. Insets on the right depict magnified images of the same surface at 7.5 min. (c) Time courses of the mean Atto647N-tubulin fluorescence intensities measured at the surface for the entire field of view for different chTOG, TPX2 and Kin1 rigor densities. Concentrations of biotinylated proteins used for surface incubation as indicated. The same Kin1 rigor curve is shown as a control for both chTOG and TPX2 plots. Note that the fluorescence signal typically represents the sum of several different tubulin species: soluble tubulin in the TIRF field (creating part of the background), immobilised individual tubulins (e.g. bound to chTOG), tubulin ‘stubs’ (bound to TPX2) and microtubules (bound to chTOG or TPX2). Raw intensities including background are shown. (d) Time series of TIRF microscopy images showing nucleation and growth of Atto647N-labelled microtubules on surfaces with immobilised biotinylated TPX2 (pre-incubated at 125 nM, top three rows) or, for controls, with a biotinylated Kin1 rigor (pre-incubated at 125 nM, bottom two rows) in either the absence or presence of 100 nM untagged chTOG, as indicated. Because of the high microtubule density at the TPX2 surface in the presence of chTOG, the same time series is also shown with reduced contrast for better visualisation of individual microtubules. Atto647N-labelled tubulin concentration was always 12.5 μM. Scale bars as indicated. t = 0 when the sample is placed at 30°C.

Techniques Used: Microscopy, Mutagenesis, Incubation, Fluorescence, Concentration Assay

Regulation of TPX2 and chTOG-stimulated microtubule nucleation by importins (a) Time series of TIRF microscopy images showing microtubule nucleation and growth on surfaces with immobilised biotinylated rigor kinesin (pre-incubated at 125 nM) always in the presence of 100 nM chTOG and Atto647N-labelled tubulin, without and with additional 500 nM importin α/ β complex (first and second row, respectively), 100 nM mGFP-TPX2 (third row) or both 500 nM importin α/β and 100 nM mGFP-TPX2 (fourth row) as indicated. Plots showing the time course of the mean Atto647N-labelled microtubule (magenta) and mGFP-TPX2 (green) intensities for the same experiments are shown next to each individual times series of images. Note that the mGFP-TPX2 signal declines at later time points likely due to depletion of TPX2 from solution due to binding to the efficiently nucleated growing microtubules. (b) Time series of TIRF microscopy images showing that compared to a control (left), inclusion of importin α/β (500 nM) inhibits stabilisation of Atto647N-labelled nucleation intermediates by surface-immobilised biotinylated TPX2 (pre-incubated at 125 nM) (right image pair). Plot showing the time course of the mean Atto647N-labelled tubulin intensities on the surface (right). Atto647N-labelled tubulin concentration was always 12.5 μM. Scale bars as indicated. t = 0 when the sample is placed at 30°C. (c) Model of synergistic TPX2 and chTOG-stimulated microtubule nucleation and growth. Different reactions, all promoting nucleation and growth, are regulated by the distinct and complementary activities of TPX2 and chTOG.
Figure Legend Snippet: Regulation of TPX2 and chTOG-stimulated microtubule nucleation by importins (a) Time series of TIRF microscopy images showing microtubule nucleation and growth on surfaces with immobilised biotinylated rigor kinesin (pre-incubated at 125 nM) always in the presence of 100 nM chTOG and Atto647N-labelled tubulin, without and with additional 500 nM importin α/ β complex (first and second row, respectively), 100 nM mGFP-TPX2 (third row) or both 500 nM importin α/β and 100 nM mGFP-TPX2 (fourth row) as indicated. Plots showing the time course of the mean Atto647N-labelled microtubule (magenta) and mGFP-TPX2 (green) intensities for the same experiments are shown next to each individual times series of images. Note that the mGFP-TPX2 signal declines at later time points likely due to depletion of TPX2 from solution due to binding to the efficiently nucleated growing microtubules. (b) Time series of TIRF microscopy images showing that compared to a control (left), inclusion of importin α/β (500 nM) inhibits stabilisation of Atto647N-labelled nucleation intermediates by surface-immobilised biotinylated TPX2 (pre-incubated at 125 nM) (right image pair). Plot showing the time course of the mean Atto647N-labelled tubulin intensities on the surface (right). Atto647N-labelled tubulin concentration was always 12.5 μM. Scale bars as indicated. t = 0 when the sample is placed at 30°C. (c) Model of synergistic TPX2 and chTOG-stimulated microtubule nucleation and growth. Different reactions, all promoting nucleation and growth, are regulated by the distinct and complementary activities of TPX2 and chTOG.

Techniques Used: Microscopy, Incubation, Binding Assay, Concentration Assay

The central region of TPX2 is sufficient to stimulate microtubule nucleation (a) Times series of TIRF microscopy images of the ‘solution’ nucleation assay where microtubules that nucleated in solution in the presence of different concentrations of biotinylated TPX2 mini and in the additional presence of untagged chTOG, followed after 1 min by binding to a neutravidin-coated surface. (b) Time series of TIRF microscopy images of the ‘surface’ nucleation experiments with immobilised biotinylated TPX2 mini (pre-incubated at 500 nM) in the absence (top row) or presence (bottom row) of untagged chTOG. Atto647N-labelled tubulin concentration was always 12.5 μM. Other protein concentrations and scale bars as indicated. t = 0 when the sample is placed at 30°C.
Figure Legend Snippet: The central region of TPX2 is sufficient to stimulate microtubule nucleation (a) Times series of TIRF microscopy images of the ‘solution’ nucleation assay where microtubules that nucleated in solution in the presence of different concentrations of biotinylated TPX2 mini and in the additional presence of untagged chTOG, followed after 1 min by binding to a neutravidin-coated surface. (b) Time series of TIRF microscopy images of the ‘surface’ nucleation experiments with immobilised biotinylated TPX2 mini (pre-incubated at 500 nM) in the absence (top row) or presence (bottom row) of untagged chTOG. Atto647N-labelled tubulin concentration was always 12.5 μM. Other protein concentrations and scale bars as indicated. t = 0 when the sample is placed at 30°C.

Techniques Used: Microscopy, Binding Assay, Incubation, Concentration Assay

The central part of human TPX2 determines its binding preference for growing microtubule ends (a) Scheme of the human TPX2 constructs used in this study: full-length TPX2, N-terminally truncated TPX2 containing amino acids 274 - 747 (TPX2 ΔN ), and a minimal TPX2 construct containing amino acids 274 - 659 (TPX2 mini ). Regions known to interact with Aurora A (AurA), importin α (Imp α) are indicated, together with predicted coil coils (CC) and nuclear localisation signal (NLS). (b) Coomassie Blue-stained SDS-PAGE gel showing 1 μg of purified recombinant TPX2 constructs. (c, d) Single channel and merged TIRF microscopy images showing how mGFP-TPX2 (green in merge) at 5 nM (c) and 0.35 nM (d) binds either all along (c) or preferentially to the growing ends and the GMPCPP segment (d) of a growing Atto647N-labelled microtubule (magenta in merge) (“−“ and “+” indicate end binding, the GMPCPP “seed” is marked by an arrowhead). (e) Kymographs depicting the time course of binding of 5 nM mGFP-TPX2 all along a growing microtubule. Att647N-labelled tubulin concentration was 7.5 μM. (f) Kymographs depicting the time course of 0.35 nM mGFP-TPX2 binding to a growing microtubule end and the GMPCPP “seed”. Att647N-labelled tubulin concentration was 12.5 μM. (g, h) Kymographs showing binding of (g) 10 nM mGFP-TPX2 ΔN and (h) 33 nM mGFP-TPX2 mini to dynamic microtubules (merged channels on the left, mGFP-TPX2 on the right). Atto647N-labelled tubulin concentration is 12.5 and 15 μM, respectively. (i, j) Kymographs showing that neither (i) 1 nM full-length mGFP-TPX2 nor (j) 10 nM TPX2 mini binds to shrinking microtubules. Atto647N-labelled tubulin concentrations were 5 μM and 7.5 μM, respectively. For all kymograph pairs: merged channel - left, mGFP-TPX2 channel - right. Scale bars as indicated.
Figure Legend Snippet: The central part of human TPX2 determines its binding preference for growing microtubule ends (a) Scheme of the human TPX2 constructs used in this study: full-length TPX2, N-terminally truncated TPX2 containing amino acids 274 - 747 (TPX2 ΔN ), and a minimal TPX2 construct containing amino acids 274 - 659 (TPX2 mini ). Regions known to interact with Aurora A (AurA), importin α (Imp α) are indicated, together with predicted coil coils (CC) and nuclear localisation signal (NLS). (b) Coomassie Blue-stained SDS-PAGE gel showing 1 μg of purified recombinant TPX2 constructs. (c, d) Single channel and merged TIRF microscopy images showing how mGFP-TPX2 (green in merge) at 5 nM (c) and 0.35 nM (d) binds either all along (c) or preferentially to the growing ends and the GMPCPP segment (d) of a growing Atto647N-labelled microtubule (magenta in merge) (“−“ and “+” indicate end binding, the GMPCPP “seed” is marked by an arrowhead). (e) Kymographs depicting the time course of binding of 5 nM mGFP-TPX2 all along a growing microtubule. Att647N-labelled tubulin concentration was 7.5 μM. (f) Kymographs depicting the time course of 0.35 nM mGFP-TPX2 binding to a growing microtubule end and the GMPCPP “seed”. Att647N-labelled tubulin concentration was 12.5 μM. (g, h) Kymographs showing binding of (g) 10 nM mGFP-TPX2 ΔN and (h) 33 nM mGFP-TPX2 mini to dynamic microtubules (merged channels on the left, mGFP-TPX2 on the right). Atto647N-labelled tubulin concentration is 12.5 and 15 μM, respectively. (i, j) Kymographs showing that neither (i) 1 nM full-length mGFP-TPX2 nor (j) 10 nM TPX2 mini binds to shrinking microtubules. Atto647N-labelled tubulin concentrations were 5 μM and 7.5 μM, respectively. For all kymograph pairs: merged channel - left, mGFP-TPX2 channel - right. Scale bars as indicated.

Techniques Used: Binding Assay, Construct, Staining, SDS Page, Purification, Recombinant, Microscopy, Concentration Assay

Effect of full-length TPX2 and TPX2 mini on microtubule dynamic instability parameters (a) Modified box-and-whiskers graph for the microtubule growth speeds in the absence (control) and presence of different concentrations of full-length mGFP-TPX2 and mGFP-TPX2 mini , as indicated. Number of observed microtubule growth episodes per condition: control – n=150; mGFP-TPX2: 0.2 nM – n= 160, 1 nM – n=150, 5 nM – n=114; mGFP-TPX2 mini : 10 nM – n=153, 50 nM – n=121, 250 nM – n=154. All events are from one dataset each. (b) TIRF microscopy kymographs showing dynamic Atto647N-labelled microtubules in the absence (control) or presence of 5 nM mGFP-TPX2 or 250 nM mGFP-TPX2 mini . Scale bars as indicated. Bar graphs showing microtubule (c) catastrophe and (d) rescue frequencies, and box-and-whiskers graphs showing microtubule (e) growth and (f) depolymerisation speeds, for control, 5 nM mGFP-TPX2, and 250 nM mGFP-TPX2 mini , as indicated. Box-and-whiskers graphs show (g) microtubule lifetimes, (h) depolymerisation times and (i) depolymerisation lengths for the same conditions. Data for (c-i) were pooled from three datasets each. Total number of analysed events per condition: (c) catastrophes (total growth time in brackets): control – n=757 (457,280 s), mGFP-TPX2 –n= 209 (267,644 s), mGFP-TPX2 mini – n=385 (409,675 s); (d) rescues (total depolymerisation time in brackets): control – n=612 (22,062 s), mGFP-TPX2 – n=209 (10,708 s), mGFP-TPX2 mini – n=376 (13,130 s); (e) growth episodes: control – n=863, 5 nM mGFP-TPX2 – n=271, 250 nM mGFP-TPX2 mini –n=477; (f) depolymerisation episodes: control – n=756, 5 nM, mGFP-TPX2 – n=211, 250 nM mGFP-TPX2 mini –n=383. The same data were used in (g)-(i) (see Supplementary Note ). Atto647N-labelled tubulin concentration was always 7.5 μM. Errors in bar graphs are SEM. For the modified box-and-whiskers plots the boxes range from 25 th to 75 th percentile, the whiskers span from 10 th to 90 th percentile, the horizontal line marks the mean value. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 (only displayed for comparisons with control); determined for the comparison of mean values analysing raw data (Tukey’s test in conjunction with One Way ANOVA).
Figure Legend Snippet: Effect of full-length TPX2 and TPX2 mini on microtubule dynamic instability parameters (a) Modified box-and-whiskers graph for the microtubule growth speeds in the absence (control) and presence of different concentrations of full-length mGFP-TPX2 and mGFP-TPX2 mini , as indicated. Number of observed microtubule growth episodes per condition: control – n=150; mGFP-TPX2: 0.2 nM – n= 160, 1 nM – n=150, 5 nM – n=114; mGFP-TPX2 mini : 10 nM – n=153, 50 nM – n=121, 250 nM – n=154. All events are from one dataset each. (b) TIRF microscopy kymographs showing dynamic Atto647N-labelled microtubules in the absence (control) or presence of 5 nM mGFP-TPX2 or 250 nM mGFP-TPX2 mini . Scale bars as indicated. Bar graphs showing microtubule (c) catastrophe and (d) rescue frequencies, and box-and-whiskers graphs showing microtubule (e) growth and (f) depolymerisation speeds, for control, 5 nM mGFP-TPX2, and 250 nM mGFP-TPX2 mini , as indicated. Box-and-whiskers graphs show (g) microtubule lifetimes, (h) depolymerisation times and (i) depolymerisation lengths for the same conditions. Data for (c-i) were pooled from three datasets each. Total number of analysed events per condition: (c) catastrophes (total growth time in brackets): control – n=757 (457,280 s), mGFP-TPX2 –n= 209 (267,644 s), mGFP-TPX2 mini – n=385 (409,675 s); (d) rescues (total depolymerisation time in brackets): control – n=612 (22,062 s), mGFP-TPX2 – n=209 (10,708 s), mGFP-TPX2 mini – n=376 (13,130 s); (e) growth episodes: control – n=863, 5 nM mGFP-TPX2 – n=271, 250 nM mGFP-TPX2 mini –n=477; (f) depolymerisation episodes: control – n=756, 5 nM, mGFP-TPX2 – n=211, 250 nM mGFP-TPX2 mini –n=383. The same data were used in (g)-(i) (see Supplementary Note ). Atto647N-labelled tubulin concentration was always 7.5 μM. Errors in bar graphs are SEM. For the modified box-and-whiskers plots the boxes range from 25 th to 75 th percentile, the whiskers span from 10 th to 90 th percentile, the horizontal line marks the mean value. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 (only displayed for comparisons with control); determined for the comparison of mean values analysing raw data (Tukey’s test in conjunction with One Way ANOVA).

Techniques Used: Modification, Microscopy, Concentration Assay

TPX2 binds to a unique binding region at growing microtubule ends (a) Right: Images of 33 nM mGFP-TPX2 mini (green in merge) at growing Atto647N-labelled microtubule ends (magenta in merge) at the indicated tubulin concentrations. Left: Averaged mGFP-TPX2 mini intensity profiles at the end regions of microtubules growing in the presence of 33 nM mGFP-TPX2 mini and the indicated Atto647N-labelled tubulin concentrations. (b) Averaged mGFP-TPX2 mini intensity profiles at the end regions of microtubules growing in the presence of 33 nM mGFP-TPX2 mini and 20 μM Atto647N-labelled tubulin for three different time windows after start of growth. (c) Averaged mGFP-TPX2 mini intensity profiles for the indicated mGFP-TPX2 mini concentrations at 25 μM Atto647N-labelled tubulin. (d) Cartoons depicting the assay conditions and TIRF microscopy kymographs showing single molecule binding events of 5 nM mGFP-TPX2 mini (green in merge) to the ends of growing Atto565-labelled microtubules (blue in merge) in the absence or presence of 195 nM Alexa647-labelled SNAP-TPX2 mini (magenta in merge). (e) Dissociation rate constant k off and association rate r on , respectively, as determined for the conditions in (d) from the dwell and waiting time distributions of the single mGFP-TPX2 mini molecule binding and unbinding events ( Supplementary Fig. 3a-c , Supplementary Note ) as a function of the distance from the growing microtubule plus end. Note that the increase in k off values and decrease in r on values is less steep in the presence of excess Alexa647-labelled SNAP-TPX2 mini , agreeing with (4c). Lower panel: The normalised total fluorescence intensity of all mGFP-TPX2 mini binding events shows their steady state distribution. (f) Kymographs and instantaneous intensity line scans at different times, as indicated, showing strong temporal fluctuations of the fluorescence intensity of mGFP-TPX2 mini (25 nM, green in merge) at growing Atto647N-labelled microtubule ends (magenta in merge, 12.5 μM tubulin). (g) TIRF microscopy images showing 33 nM mGFP-TPX2 mini (green in merge, lower panels) accumulating strongly at the curved microtubule ends of Atto647N-labelled growing microtubules (magenta in merge, 30 μM tubulin). (h) Kymographs of 5 nM mGFP-TPX2 (green in merge) localising to the ends of Atto647N-labelled microtubules (magenta in merge, 25 μM tubulin) growing in the presence of the slowly-hydrolysable GTP analogue GTPγS. Scale bars as indicated.
Figure Legend Snippet: TPX2 binds to a unique binding region at growing microtubule ends (a) Right: Images of 33 nM mGFP-TPX2 mini (green in merge) at growing Atto647N-labelled microtubule ends (magenta in merge) at the indicated tubulin concentrations. Left: Averaged mGFP-TPX2 mini intensity profiles at the end regions of microtubules growing in the presence of 33 nM mGFP-TPX2 mini and the indicated Atto647N-labelled tubulin concentrations. (b) Averaged mGFP-TPX2 mini intensity profiles at the end regions of microtubules growing in the presence of 33 nM mGFP-TPX2 mini and 20 μM Atto647N-labelled tubulin for three different time windows after start of growth. (c) Averaged mGFP-TPX2 mini intensity profiles for the indicated mGFP-TPX2 mini concentrations at 25 μM Atto647N-labelled tubulin. (d) Cartoons depicting the assay conditions and TIRF microscopy kymographs showing single molecule binding events of 5 nM mGFP-TPX2 mini (green in merge) to the ends of growing Atto565-labelled microtubules (blue in merge) in the absence or presence of 195 nM Alexa647-labelled SNAP-TPX2 mini (magenta in merge). (e) Dissociation rate constant k off and association rate r on , respectively, as determined for the conditions in (d) from the dwell and waiting time distributions of the single mGFP-TPX2 mini molecule binding and unbinding events ( Supplementary Fig. 3a-c , Supplementary Note ) as a function of the distance from the growing microtubule plus end. Note that the increase in k off values and decrease in r on values is less steep in the presence of excess Alexa647-labelled SNAP-TPX2 mini , agreeing with (4c). Lower panel: The normalised total fluorescence intensity of all mGFP-TPX2 mini binding events shows their steady state distribution. (f) Kymographs and instantaneous intensity line scans at different times, as indicated, showing strong temporal fluctuations of the fluorescence intensity of mGFP-TPX2 mini (25 nM, green in merge) at growing Atto647N-labelled microtubule ends (magenta in merge, 12.5 μM tubulin). (g) TIRF microscopy images showing 33 nM mGFP-TPX2 mini (green in merge, lower panels) accumulating strongly at the curved microtubule ends of Atto647N-labelled growing microtubules (magenta in merge, 30 μM tubulin). (h) Kymographs of 5 nM mGFP-TPX2 (green in merge) localising to the ends of Atto647N-labelled microtubules (magenta in merge, 25 μM tubulin) growing in the presence of the slowly-hydrolysable GTP analogue GTPγS. Scale bars as indicated.

Techniques Used: Binding Assay, Microscopy, Fluorescence

In solution TPX2 nucleates microtubules more efficiently than chTOG (a) Scheme of the experimental setup for the ‘solution’ nucleation assay. (b) Time series of TIRF microscopy images showing Atto647N-labelled microtubules that nucleated in solution in the presence of nucleation factors as indicated, followed after 1 min by binding to a neutravidin-coated surface via the biotinylated protein present as indicated. (c) TIRF microscopy images showing examples the concentration dependent nucleation efficiencies of chTOG- and TPX2-mediated microtubule nucleation in the ‘solution’ nucleation assay. (d) Time courses of mean Atto647N-tubulin fluorescence intensities measured for the entire field of view in ‘solution’ at the biotinylated chTOG and TPX2 concentrations as indicated. Note that the fluorescence signal typically represents the sum of several different tubulin species (see note in legend of Fig. 5c ). Raw intensities including background are shown. Atto647N-labelled tubulin concentration was always 12.5 μM. Other protein concentrations and scale bar as indicated. t = 0 when the sample is placed at 30°C.
Figure Legend Snippet: In solution TPX2 nucleates microtubules more efficiently than chTOG (a) Scheme of the experimental setup for the ‘solution’ nucleation assay. (b) Time series of TIRF microscopy images showing Atto647N-labelled microtubules that nucleated in solution in the presence of nucleation factors as indicated, followed after 1 min by binding to a neutravidin-coated surface via the biotinylated protein present as indicated. (c) TIRF microscopy images showing examples the concentration dependent nucleation efficiencies of chTOG- and TPX2-mediated microtubule nucleation in the ‘solution’ nucleation assay. (d) Time courses of mean Atto647N-tubulin fluorescence intensities measured for the entire field of view in ‘solution’ at the biotinylated chTOG and TPX2 concentrations as indicated. Note that the fluorescence signal typically represents the sum of several different tubulin species (see note in legend of Fig. 5c ). Raw intensities including background are shown. Atto647N-labelled tubulin concentration was always 12.5 μM. Other protein concentrations and scale bar as indicated. t = 0 when the sample is placed at 30°C.

Techniques Used: Microscopy, Binding Assay, Concentration Assay, Fluorescence

22) Product Images from "Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages"

Article Title: Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

Journal: Journal of Virology

doi: 10.1128/JVI.75.22.11088-11095.2001

Effects of pH (A, D, and G) and of ammonium acetate (B, E, and H) and Mn 2+ (C, F, and I) concentrations on the activities of the three polymerases. Reaction mixtures containing s + 13 RNA and φ6 Pol (A, B, and C), φ8 Pol (D, E, and F) or φ13 Pol (G, H, and I) were incubated at 30°C for 1 h and analyzed by agarose gel electrophoresis. Radioactivity in the bands of the newly produced dsRNA was quantified with a phosphorimager. Tris-HCl (Tris) or HEPES-KOH (HEPES) buffers were used in panelsA, D, and G, whereas in the other panels pH was buffered with HEPES-KOH (pH 7.8 for φ6 Pol and φ8 Pol, and pH 7.4 for φ13 Pol). Graphs are normalized so that the highest observed value within each panel is set to 1.
Figure Legend Snippet: Effects of pH (A, D, and G) and of ammonium acetate (B, E, and H) and Mn 2+ (C, F, and I) concentrations on the activities of the three polymerases. Reaction mixtures containing s + 13 RNA and φ6 Pol (A, B, and C), φ8 Pol (D, E, and F) or φ13 Pol (G, H, and I) were incubated at 30°C for 1 h and analyzed by agarose gel electrophoresis. Radioactivity in the bands of the newly produced dsRNA was quantified with a phosphorimager. Tris-HCl (Tris) or HEPES-KOH (HEPES) buffers were used in panelsA, D, and G, whereas in the other panels pH was buffered with HEPES-KOH (pH 7.8 for φ6 Pol and φ8 Pol, and pH 7.4 for φ13 Pol). Graphs are normalized so that the highest observed value within each panel is set to 1.

Techniques Used: Incubation, Agarose Gel Electrophoresis, Radioactivity, Produced

23) Product Images from "Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol"

Article Title: Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol

Journal: PLoS ONE

doi: 10.1371/journal.pone.0184489

The reaction buffer composition affects the DNA polymerase activity of PrimPol in vitro . (A) the DNA polymerase activity of PrimPol at different NaCl concentrations. (B) the DNA polymerase activity of PrimPol at different pH. HEPES based buffer was used to test the DNA polymerase activity of PrimPol at different pH values. (C) the DNA polymerase activity of PrimPol at different MgCl 2 and MnCl 2 concentrations. 200 nM of PrimPol and 25 nM 70-mer DNA template were used in the reactions. All experiments were repeated three times.
Figure Legend Snippet: The reaction buffer composition affects the DNA polymerase activity of PrimPol in vitro . (A) the DNA polymerase activity of PrimPol at different NaCl concentrations. (B) the DNA polymerase activity of PrimPol at different pH. HEPES based buffer was used to test the DNA polymerase activity of PrimPol at different pH values. (C) the DNA polymerase activity of PrimPol at different MgCl 2 and MnCl 2 concentrations. 200 nM of PrimPol and 25 nM 70-mer DNA template were used in the reactions. All experiments were repeated three times.

Techniques Used: Activity Assay, In Vitro

24) Product Images from "The electron distribution in the “activated” state of cytochrome c oxidase"

Article Title: The electron distribution in the “activated” state of cytochrome c oxidase

Journal: Scientific Reports

doi: 10.1038/s41598-018-25779-w

Absorbance difference spectra. A comparison of reduced minus oxidized difference spectra of the Cyt c O-cyt. c complex (black) and the sum of the reduced minus oxidized difference spectra of Cyt c O and cyt. c (red), respectively. Spectra of the oxidized states were recorded first and then the atmosphere in the cuvette was replaced for N 2 after which the samples were reduced with ascorbate (2 mM) and hexaammineruthenium(II) chloride (1 μM). Reduction of the samples was followed in time by recording spectra over ~2 hours until no further changes were observed. The data to the right of the axis break have been multiplied by a factor of five. The concentrations of Cyt c O and cyt. c were ~3 μM and ~5 μM, respectively, in 50 mM HEPES (pH 7.5), 0.05% DDM and 100 µM EDTA.
Figure Legend Snippet: Absorbance difference spectra. A comparison of reduced minus oxidized difference spectra of the Cyt c O-cyt. c complex (black) and the sum of the reduced minus oxidized difference spectra of Cyt c O and cyt. c (red), respectively. Spectra of the oxidized states were recorded first and then the atmosphere in the cuvette was replaced for N 2 after which the samples were reduced with ascorbate (2 mM) and hexaammineruthenium(II) chloride (1 μM). Reduction of the samples was followed in time by recording spectra over ~2 hours until no further changes were observed. The data to the right of the axis break have been multiplied by a factor of five. The concentrations of Cyt c O and cyt. c were ~3 μM and ~5 μM, respectively, in 50 mM HEPES (pH 7.5), 0.05% DDM and 100 µM EDTA.

Techniques Used:

25) Product Images from "HA95 and LAP2? mediate a novel chromatin-nuclear envelope interaction implicated in initiation of DNA replication"

Article Title: HA95 and LAP2? mediate a novel chromatin-nuclear envelope interaction implicated in initiation of DNA replication

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200210026

LAP2β(137–298) inhibits initiation of DNA replication in G1 nuclei. (A) Peptide-loaded G1 nuclei were incubated in S-phase extract containing [α 32 P]dCTP, dNTPs, GTP, and an ATP-regenerating system. [α 32 P]dCTP incorporation was analyzed by phosphorImager (cpm/10 5 nuclei). (B) Nuclei isolated from HeLa cells at the indicated cell cycle stages were incubated in extracts from S or G0 cells under conditions promoting replication. S-phase extracts also contained 1 mM olomoucine (+Olo) or H 2 O (−Olo) (right panel). Synthesized DNA was analyzed by autoradiography. (C) Relative BAF content of G1 nuclei loaded with the indicated peptides and exposed to S-phase extract was examined by immunoblotting. HA95 was also detected as a loading control in the gel. (D) Summary of GST–LAP2β peptides harboring HA95-NBD supporting (+) or inhibiting (−) replication in G1 nuclei. (E) BrdU density substitution experiment. G1 nuclei containing either no peptide (circles), GST alone (triangles), or GST–LAP2β(137–298) (squares) were allowed to replicate in S-phase extract containing BrdU and [α 32 P]dCTP. Substituted DNA was separated by centrifugation in CsCl gradients. cpm of each fraction was plotted against fractions of equal densities. HL indicates density of heavy-light replicated DNA. (F) Nuclei from S-phase HeLa cells were loaded with GST–LAP2β peptides and peptide uptake was analyzed as in Fig. 4 A (GST–LAP2β[1–452] is shown). Peptide-loaded nuclei were incubated in S-phase extract and incorporation of [α 32 P]dCTP was measured by phosphorImager (cpm/10 5 nuclei). Bar, 10 μM.
Figure Legend Snippet: LAP2β(137–298) inhibits initiation of DNA replication in G1 nuclei. (A) Peptide-loaded G1 nuclei were incubated in S-phase extract containing [α 32 P]dCTP, dNTPs, GTP, and an ATP-regenerating system. [α 32 P]dCTP incorporation was analyzed by phosphorImager (cpm/10 5 nuclei). (B) Nuclei isolated from HeLa cells at the indicated cell cycle stages were incubated in extracts from S or G0 cells under conditions promoting replication. S-phase extracts also contained 1 mM olomoucine (+Olo) or H 2 O (−Olo) (right panel). Synthesized DNA was analyzed by autoradiography. (C) Relative BAF content of G1 nuclei loaded with the indicated peptides and exposed to S-phase extract was examined by immunoblotting. HA95 was also detected as a loading control in the gel. (D) Summary of GST–LAP2β peptides harboring HA95-NBD supporting (+) or inhibiting (−) replication in G1 nuclei. (E) BrdU density substitution experiment. G1 nuclei containing either no peptide (circles), GST alone (triangles), or GST–LAP2β(137–298) (squares) were allowed to replicate in S-phase extract containing BrdU and [α 32 P]dCTP. Substituted DNA was separated by centrifugation in CsCl gradients. cpm of each fraction was plotted against fractions of equal densities. HL indicates density of heavy-light replicated DNA. (F) Nuclei from S-phase HeLa cells were loaded with GST–LAP2β peptides and peptide uptake was analyzed as in Fig. 4 A (GST–LAP2β[1–452] is shown). Peptide-loaded nuclei were incubated in S-phase extract and incorporation of [α 32 P]dCTP was measured by phosphorImager (cpm/10 5 nuclei). Bar, 10 μM.

Techniques Used: Incubation, Isolation, Synthesized, Autoradiography, Centrifugation

26) Product Images from "Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly"

Article Title: Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.2932

Binding of M. jannaschii FlaH to immobilized ATP. 10 μg of FlaH were incubated with 20 μL of ATP‐agarose in 20 m M HEPES, pH 8.0, 100 m M NaCl, 5 m M MgCl 2 for 2 h at 30°C. Samples were analyzed by SDS‐PAGE. 1, loaded protein; 2, supernatant after incubation; 3, ATP elution; 4, elution with SDS‐PAAG loading buffer. Agarose was used as a blank control.
Figure Legend Snippet: Binding of M. jannaschii FlaH to immobilized ATP. 10 μg of FlaH were incubated with 20 μL of ATP‐agarose in 20 m M HEPES, pH 8.0, 100 m M NaCl, 5 m M MgCl 2 for 2 h at 30°C. Samples were analyzed by SDS‐PAGE. 1, loaded protein; 2, supernatant after incubation; 3, ATP elution; 4, elution with SDS‐PAAG loading buffer. Agarose was used as a blank control.

Techniques Used: Binding Assay, Incubation, SDS Page

27) Product Images from "A Novel Mammalian Flavin-dependent Histone Demethylase *"

Article Title: A Novel Mammalian Flavin-dependent Histone Demethylase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.003087

Absorption spectra of LSD2 (10 μ m protein in 50 m m Hepes/NaOH, pH 8.0, 5% (w/v) glycerol) in the native oxidized state ( black line ) and after incubation with an excess of the inhibitor tranylcypromine ( gray line ). The oxidized enzyme exhibits
Figure Legend Snippet: Absorption spectra of LSD2 (10 μ m protein in 50 m m Hepes/NaOH, pH 8.0, 5% (w/v) glycerol) in the native oxidized state ( black line ) and after incubation with an excess of the inhibitor tranylcypromine ( gray line ). The oxidized enzyme exhibits

Techniques Used: Incubation

28) Product Images from "Anti-Double-Stranded DNA IgG Participates in Renal Fibrosis through Suppressing the Suppressor of Cytokine Signaling 1 Signals"

Article Title: Anti-Double-Stranded DNA IgG Participates in Renal Fibrosis through Suppressing the Suppressor of Cytokine Signaling 1 Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00610

The interactions between anti-dsDNA IgG and suppressor of cytokine signaling 1 (SOCS1) [or kinase inhibitory region (KIR) peptide] . (A) By immunoprecipitation and Western blotting, the lysate proteins in mesangial cells were selected by anti-DNA IgG and then detected with anti-SOCS1 antibody. (B) By inhibition enzyme-linked immunosorbent assay (ELISA), the binding of anti-dsDNA IgG to double-stranded DNA (dsDNA) antigen was measured upon addition of KIR (or scrambled) peptide. (C) The binding of anti-dsDNA IgG to biotinilyated KIR peptide was measured by direct ELISA. (D) Surface plasmon resonance was performed to quantitate the affinity of anti-dsDNA IgG to KIR peptide. Anti-dsDNA IgG was immobilized to sensor chip, followed by running KIR peptide (0–250 nM). Ka (1/Ms) = 3.65 × 10 4 , Kd (1/s) = 4 × 10 −3 , K D (M) = 1.1 × 10 −7 , and Rmax (RU) = 351. (E) The catalytic effect of anti-dsDNA IgG on KIR peptide was detected by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. Data were from three independent experiments. Data points and error bars represent mean ± SEM. Representative graphs are shown.
Figure Legend Snippet: The interactions between anti-dsDNA IgG and suppressor of cytokine signaling 1 (SOCS1) [or kinase inhibitory region (KIR) peptide] . (A) By immunoprecipitation and Western blotting, the lysate proteins in mesangial cells were selected by anti-DNA IgG and then detected with anti-SOCS1 antibody. (B) By inhibition enzyme-linked immunosorbent assay (ELISA), the binding of anti-dsDNA IgG to double-stranded DNA (dsDNA) antigen was measured upon addition of KIR (or scrambled) peptide. (C) The binding of anti-dsDNA IgG to biotinilyated KIR peptide was measured by direct ELISA. (D) Surface plasmon resonance was performed to quantitate the affinity of anti-dsDNA IgG to KIR peptide. Anti-dsDNA IgG was immobilized to sensor chip, followed by running KIR peptide (0–250 nM). Ka (1/Ms) = 3.65 × 10 4 , Kd (1/s) = 4 × 10 −3 , K D (M) = 1.1 × 10 −7 , and Rmax (RU) = 351. (E) The catalytic effect of anti-dsDNA IgG on KIR peptide was detected by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. Data were from three independent experiments. Data points and error bars represent mean ± SEM. Representative graphs are shown.

Techniques Used: Immunoprecipitation, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay, Direct ELISA, SPR Assay, Chromatin Immunoprecipitation, Mass Spectrometry

The effect of d -form ALW peptide on anti-dsDNA IgG binding to double-stranded DNA (dsDNA) antigen or kinase inhibitory region (KIR) peptide . (A) The catalytic effect of anti-dsDNA IgG on d -form or l -form ALW peptide was detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. (B) Inhibition enzyme-linked immunosorbent assay (ELISA) was performed for the binding of anti-dsDNA IgG to dsDNA antigen upon the addition of ALW peptides. (C) Similarly, inhibition ELISA was performed for the binding of anti-dsDNA IgG to suppressor of cytokine signaling 1 (SOCS1)-KIR peptide upon the addition of d -form or scrambled ALW peptide. Data were from three independent experiments. Data points and error bars represent mean ± SEM. Representative graphs are shown.
Figure Legend Snippet: The effect of d -form ALW peptide on anti-dsDNA IgG binding to double-stranded DNA (dsDNA) antigen or kinase inhibitory region (KIR) peptide . (A) The catalytic effect of anti-dsDNA IgG on d -form or l -form ALW peptide was detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. (B) Inhibition enzyme-linked immunosorbent assay (ELISA) was performed for the binding of anti-dsDNA IgG to dsDNA antigen upon the addition of ALW peptides. (C) Similarly, inhibition ELISA was performed for the binding of anti-dsDNA IgG to suppressor of cytokine signaling 1 (SOCS1)-KIR peptide upon the addition of d -form or scrambled ALW peptide. Data were from three independent experiments. Data points and error bars represent mean ± SEM. Representative graphs are shown.

Techniques Used: Binding Assay, Mass Spectrometry, Inhibition, Enzyme-linked Immunosorbent Assay

The interactions between the suppressor of cytokine signaling 1 (SOCS1) kinase inhibitory region (KIR) and Janus kinase 2 (JAK2) loop peptides . (A) Surface plasmon resonance was performed for the binding affinity of JAK2 loop peptide to biotinylated KIR peptide, which was immobilized on sensor chip. JAK2 loop peptide was run at a concentration of 0–250 nM. Ka (1/Ms) = 1.35 × 10 4 , Kd (1/s) = 6.03 × 10 −4 , K D (M) = 4.46 × 10 −8 , and Rmax (RU) = 359. (B) By inhibition enzyme-linked immunosorbent assay (ELISA), the binding of fluorescein isothiocyanate (FITC)–JAK2 loop peptide to biotinylated KIR peptide was measured upon the addition of control or anti-dsDNA IgG. Data were from three independent experiments. Data points and error bars represent mean ± SEM. Representative graphs are shown.
Figure Legend Snippet: The interactions between the suppressor of cytokine signaling 1 (SOCS1) kinase inhibitory region (KIR) and Janus kinase 2 (JAK2) loop peptides . (A) Surface plasmon resonance was performed for the binding affinity of JAK2 loop peptide to biotinylated KIR peptide, which was immobilized on sensor chip. JAK2 loop peptide was run at a concentration of 0–250 nM. Ka (1/Ms) = 1.35 × 10 4 , Kd (1/s) = 6.03 × 10 −4 , K D (M) = 4.46 × 10 −8 , and Rmax (RU) = 359. (B) By inhibition enzyme-linked immunosorbent assay (ELISA), the binding of fluorescein isothiocyanate (FITC)–JAK2 loop peptide to biotinylated KIR peptide was measured upon the addition of control or anti-dsDNA IgG. Data were from three independent experiments. Data points and error bars represent mean ± SEM. Representative graphs are shown.

Techniques Used: SPR Assay, Binding Assay, Chromatin Immunoprecipitation, Concentration Assay, Mass Spectrometry, Inhibition, Enzyme-linked Immunosorbent Assay

29) Product Images from "Tuning Ciprofloxacin Release Profiles from Liposomally Encapsulated Nanocrystalline Drug"

Article Title: Tuning Ciprofloxacin Release Profiles from Liposomally Encapsulated Nanocrystalline Drug

Journal: Pharmaceutical Research

doi: 10.1007/s11095-016-2002-5

Evaluation of the effect of freeze-thaw at −50°C on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and polysorbate 20 after freeze-thaw. The CFI formulations (at 12.5 mg/ml) were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control (no freeze-thaw, blue diamonds), CFI containing 0.05% polysorbate 20 after freeze-thaw (red squares), CFI containing 0.1% polysorbate 20 after freeze-thaw (green triangles), and CFI containing 0.2% polysorbate 20 after freeze-thaw (yellow circles). Duplicate samples were analyzed at each time point.
Figure Legend Snippet: Evaluation of the effect of freeze-thaw at −50°C on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and polysorbate 20 after freeze-thaw. The CFI formulations (at 12.5 mg/ml) were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control (no freeze-thaw, blue diamonds), CFI containing 0.05% polysorbate 20 after freeze-thaw (red squares), CFI containing 0.1% polysorbate 20 after freeze-thaw (green triangles), and CFI containing 0.2% polysorbate 20 after freeze-thaw (yellow circles). Duplicate samples were analyzed at each time point.

Techniques Used: Incubation

Evaluation of the effect of freeze-thaw in liquid nitrogen on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and 0, 0.05 or 0.1% polysorbate 20 after freeze-thaw. The CFI formulations (at 12.5 mg/ml) were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control prior to freeze-thaw ( blue diamonds ) or after freeze-thaw ( red circles ), CFI containing 0.05% polysorbate 20 after freeze-thaw ( green diamonds ), CFI containing 0.1% polysorbate 20 after freeze-thaw ( yellow triangles ). Duplicate samples were analyzed at each time point. Two simulated curves were generated by adjusting the release from the CFI sample without polysorbate 20 after freeze-thaw by assuming the addition of either 10% free drug ( green dashed line ) or 20% free drug ( yellow dashed line ) and normalizing the total drug release to 100%.
Figure Legend Snippet: Evaluation of the effect of freeze-thaw in liquid nitrogen on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and 0, 0.05 or 0.1% polysorbate 20 after freeze-thaw. The CFI formulations (at 12.5 mg/ml) were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control prior to freeze-thaw ( blue diamonds ) or after freeze-thaw ( red circles ), CFI containing 0.05% polysorbate 20 after freeze-thaw ( green diamonds ), CFI containing 0.1% polysorbate 20 after freeze-thaw ( yellow triangles ). Duplicate samples were analyzed at each time point. Two simulated curves were generated by adjusting the release from the CFI sample without polysorbate 20 after freeze-thaw by assuming the addition of either 10% free drug ( green dashed line ) or 20% free drug ( yellow dashed line ) and normalizing the total drug release to 100%.

Techniques Used: Incubation, Generated

Evaluation of the effect of freeze-thaw at −50°C on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and Brij 30 before ( a ) and after ( b ) freeze-thaw. The 12.5 mg/ml CFI formulations were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control (no freeze thaw, blue diamonds ), CFI containing 0.05% Brij 30 ( red squares ), CFI containing 0.1% Brij 30 ( green triangles ), CFI containing 0.2% Brij 30 ( orange circles ), and CFI containing 0.3% Brij 30 ( purple squares ). Duplicate samples were analyzed at each time point.
Figure Legend Snippet: Evaluation of the effect of freeze-thaw at −50°C on the IVR profiles of CFI formulations at pH 6.0 containing 90 mg/ml sucrose and Brij 30 before ( a ) and after ( b ) freeze-thaw. The 12.5 mg/ml CFI formulations were diluted to 50 μg/ml ciprofloxacin in HEPES buffered saline (HBS) prior to a 1:1 dilution in bovine serum to measure the release of ciprofloxacin after incubation at 37°C for up to 4 h. IVR profiles are shown for the CFI control (no freeze thaw, blue diamonds ), CFI containing 0.05% Brij 30 ( red squares ), CFI containing 0.1% Brij 30 ( green triangles ), CFI containing 0.2% Brij 30 ( orange circles ), and CFI containing 0.3% Brij 30 ( purple squares ). Duplicate samples were analyzed at each time point.

Techniques Used: Incubation

30) Product Images from "Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase *"

Article Title: Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.479345

P269A increases CHIP binding to Hsc70-CFTR complex. A , CFTR was synthesized in RRL and treated with hexokinase/2-deoxy glucose (− ATP ) or ATP and/or GST-CBag for 10 min. Microsomes were then isolated, solubilized, and immunoprecipitated ( IP ) with nonimmune sera ( NIS ) or anti-Hsc70 antisera. B , following synthesis, the translation reaction was incubated with hexokinase/2-deoxyglucose or GST-CBag plus ATP as in panel A . Microsomes were collected and solubilized, and CFTR was affinity-purified with immobilized His-tagged WT CHIP, P269A CHIP, or Ni-NTA ( Ni , control). C , quantification of experiments as shown in panel B (mean ± S.E. n = 3). D , microsomes were isolated after ATP depletion as in panel A and further incubated for 3 min in buffer (without ATP) or in the presence of CBag plus ATP to remove bound Hsc70, prior to affinity pulldown with His-tagged WT or P269A CHIP.
Figure Legend Snippet: P269A increases CHIP binding to Hsc70-CFTR complex. A , CFTR was synthesized in RRL and treated with hexokinase/2-deoxy glucose (− ATP ) or ATP and/or GST-CBag for 10 min. Microsomes were then isolated, solubilized, and immunoprecipitated ( IP ) with nonimmune sera ( NIS ) or anti-Hsc70 antisera. B , following synthesis, the translation reaction was incubated with hexokinase/2-deoxyglucose or GST-CBag plus ATP as in panel A . Microsomes were collected and solubilized, and CFTR was affinity-purified with immobilized His-tagged WT CHIP, P269A CHIP, or Ni-NTA ( Ni , control). C , quantification of experiments as shown in panel B (mean ± S.E. n = 3). D , microsomes were isolated after ATP depletion as in panel A and further incubated for 3 min in buffer (without ATP) or in the presence of CBag plus ATP to remove bound Hsc70, prior to affinity pulldown with His-tagged WT or P269A CHIP.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Synthesized, Isolation, Immunoprecipitation, Incubation, Affinity Purification

P269A CHIP preferentially binds ADP-Hsc70. A , Hsc70 was recovered from RRL using immobilized His-tagged WT or P269A CHIP, or Ni-NTA ( Ni ). IB , immunoblot. B , quantification of experiments as shown in panel A (mean ± S.E. n = 4). C , Hsc70 was affinity-isolated as in panel A , but using desalted RRL supplemented with ATP, ADP, or no nucleotide as indicated. Recovered Hsc70 was detected by immunoblotting. D , quantification of experiments as shown in panel C (mean ± S.E. n = 4).
Figure Legend Snippet: P269A CHIP preferentially binds ADP-Hsc70. A , Hsc70 was recovered from RRL using immobilized His-tagged WT or P269A CHIP, or Ni-NTA ( Ni ). IB , immunoblot. B , quantification of experiments as shown in panel A (mean ± S.E. n = 4). C , Hsc70 was affinity-isolated as in panel A , but using desalted RRL supplemented with ATP, ADP, or no nucleotide as indicated. Recovered Hsc70 was detected by immunoblotting. D , quantification of experiments as shown in panel C (mean ± S.E. n = 4).

Techniques Used: Chromatin Immunoprecipitation, Isolation

31) Product Images from "Remote loading of liposomes with a 124I-radioiodinated compound and their in vivo evaluation by PET/CT in a murine tumor model"

Article Title: Remote loading of liposomes with a 124I-radioiodinated compound and their in vivo evaluation by PET/CT in a murine tumor model

Journal: Theranostics

doi: 10.7150/thno.26706

Characterization of [ 124 I]ADA loaded liposomes by size exclusion column separation and radio-TLC. (A) Elution profile of [ 124 I]ADA-loaded liposomes by PD-10 column separation. The liposomes were eluted with 25 mM HEPES buffer (150 mM NaCl, pH 7.4, 310 mOsm/kg), with a loading efficiency of 77.2%. (B) Radio-TLC peak of [ 124 I]ADA after loading into liposomes.
Figure Legend Snippet: Characterization of [ 124 I]ADA loaded liposomes by size exclusion column separation and radio-TLC. (A) Elution profile of [ 124 I]ADA-loaded liposomes by PD-10 column separation. The liposomes were eluted with 25 mM HEPES buffer (150 mM NaCl, pH 7.4, 310 mOsm/kg), with a loading efficiency of 77.2%. (B) Radio-TLC peak of [ 124 I]ADA after loading into liposomes.

Techniques Used: Thin Layer Chromatography

32) Product Images from "Preventative role of interleukin-17 producing regulatory T helper type 17 (Treg17) cells in type 1 diabetes in non-obese diabetic mice"

Article Title: Preventative role of interleukin-17 producing regulatory T helper type 17 (Treg17) cells in type 1 diabetes in non-obese diabetic mice

Journal: Clinical and Experimental Immunology

doi: 10.1111/cei.12691

T helper type 17 (Th17) cells derived from BDC2·5 T cells by interleukin (IL)-23 + IL-6 polarization are highly diabetogenic, while regulatory T helper type 17 (T reg 17) cells are induced by transforming growth factor (TGF)-β + IL-6. Spleens were harvested from 6–8-week-old BDC2·5 non-obese diabetic (NOD) mice, and single-cell suspensions were prepared. The cells (2 × 10 5 cells/well) were cultured in 96-well plates in the presence of PS3 mimotope (1 µM) in complete RPMI-1640 medium at 37°C, 5% CO 2 for 5 days. Cells were either polarized with TGF-β (3 ng/ml) + IL-6 (20 ng/ml) cytokines or IL-23 (10 ng/ml) + IL-6 (20 ng/ml) cytokine cocktails. Cells stimulated with PS3 mimotope without cytokine treatment were used as a control. (a) Cells from various treatment groups were collected on day 5, washed and 5 × 10 6 cells/mouse were transferred adoptively via tail vein into 5-week-old NOD mice and the diabetes incidence was monitored. All mice injected with IL-23 + IL-6 polarized BDC2·5 cells developed diabetes. The Kaplan–Meier survival estimate was used to determine differences in the two groups. Significant difference was observed between the IL-23+IL-6 and TGF-β+IL-6 groups ( P
Figure Legend Snippet: T helper type 17 (Th17) cells derived from BDC2·5 T cells by interleukin (IL)-23 + IL-6 polarization are highly diabetogenic, while regulatory T helper type 17 (T reg 17) cells are induced by transforming growth factor (TGF)-β + IL-6. Spleens were harvested from 6–8-week-old BDC2·5 non-obese diabetic (NOD) mice, and single-cell suspensions were prepared. The cells (2 × 10 5 cells/well) were cultured in 96-well plates in the presence of PS3 mimotope (1 µM) in complete RPMI-1640 medium at 37°C, 5% CO 2 for 5 days. Cells were either polarized with TGF-β (3 ng/ml) + IL-6 (20 ng/ml) cytokines or IL-23 (10 ng/ml) + IL-6 (20 ng/ml) cytokine cocktails. Cells stimulated with PS3 mimotope without cytokine treatment were used as a control. (a) Cells from various treatment groups were collected on day 5, washed and 5 × 10 6 cells/mouse were transferred adoptively via tail vein into 5-week-old NOD mice and the diabetes incidence was monitored. All mice injected with IL-23 + IL-6 polarized BDC2·5 cells developed diabetes. The Kaplan–Meier survival estimate was used to determine differences in the two groups. Significant difference was observed between the IL-23+IL-6 and TGF-β+IL-6 groups ( P

Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Injection

Differential proliferation of polarized T helper type 17 (Th17) subsets by antigen. Spleens were harvested from 6–8-week-old BDC2·5 non-obese diabetic (NOD) mice, and single-cell suspensions were prepared. The cells (2 × 10 5 cells/well) were cultured in 96-well plates in the presence of PS3 mimotope (1 µM) as antigen in complete RPMI-1640 medium at 37°C, 5% CO 2 for 5 days. The T cells were either polarized with transforming growth factor (TGF)-β (3 ng/ml) + interleukin (IL)-6 (20 ng/ml) cytokines or IL-23 (10 ng/ml) + IL-6 (20 ng/ml) cytokine cocktails. Cells stimulated with PS3 mimotope without cytokine treatment were used as a positive control. After 72 h, [ 3 H]-thymidine was added and incorporation of thymidine was measured and reported as mean counts per minute (cpm) ± standard error of the mean (s.e.m.) and all values with P
Figure Legend Snippet: Differential proliferation of polarized T helper type 17 (Th17) subsets by antigen. Spleens were harvested from 6–8-week-old BDC2·5 non-obese diabetic (NOD) mice, and single-cell suspensions were prepared. The cells (2 × 10 5 cells/well) were cultured in 96-well plates in the presence of PS3 mimotope (1 µM) as antigen in complete RPMI-1640 medium at 37°C, 5% CO 2 for 5 days. The T cells were either polarized with transforming growth factor (TGF)-β (3 ng/ml) + interleukin (IL)-6 (20 ng/ml) cytokines or IL-23 (10 ng/ml) + IL-6 (20 ng/ml) cytokine cocktails. Cells stimulated with PS3 mimotope without cytokine treatment were used as a positive control. After 72 h, [ 3 H]-thymidine was added and incorporation of thymidine was measured and reported as mean counts per minute (cpm) ± standard error of the mean (s.e.m.) and all values with P

Techniques Used: Mouse Assay, Cell Culture, Positive Control

33) Product Images from "Spatial and temporal changes in Bax subcellular localization during anoikis"

Article Title: Spatial and temporal changes in Bax subcellular localization during anoikis

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200302154

Bax translocates to mitochondria and forms high mol wt oligomers during anoikis. (a) FSK-7 cells, adherent or detached (maintained on poly-HEMA for 15 min or 4 h), were separated into soluble (S) and membrane (M) fractions before SDS-PAGE and immunoblotting with polyclonal anti-Bax (top). 4 mM CHAPS was added, and Bax was immunoprecipitated with anti-Bax 62M. Immunoprecipitates were separated by SDS-PAGE and were immunoblotted with anti-Bax 5B7 (bottom). (b) Cells expressing the OMM marker YFP-XT were detached for various times before cytospinning, and were immunostained with the activation-dependent antibody 62M. 62M reactivity is seen at discrete regions on mitochondria at all time points. Bar, 5 μm. (c) Soluble and membrane fractions from adherent cells or cells maintained on poly-HEMA for 4 h were isolated as above. CHAPS was added to a final concentration of 4 mM, and the fractions were concentrated. 5 mg protein from each fraction was loaded onto a Sephacryl S100-HR column (see Materials and methods). Separated fractions were analyzed by SDS-PAGE and immunoblotting for Bax. (d) Membrane fractions from FSK-7 cells detached for 4 h were separated by size-exclusion chromatography as above. Fractions containing high (oligo) and low (mono) mol wt Bax complexes were collected. Fractions were concentrated and separated into three equal parts. These were treated with BS3 (with or without addition of 0.1% Triton X-100). Samples were separated by SDS-PAGE and were immunoblotted for Bax. Cytosolic Bax from adherent cells (Adh) was run for comparison after identical treatment. (e) Membrane fractions isolated from adherent cells or cells detached for various times and separated by size-exclusion chromatography were analyzed by SDS-PAGE and immunoblotting for Bax. Total soluble (S) and membrane (M) fractions show Bax translocation at each time point. (f) Membrane fractions prepared from adherent cells or cells detached for 15 min, 1 h, or 4 h were spilt into three and treated with 5 mM BS3 in the presence or absence of 0.1% Triton-X100. Samples were analyzed by SDS-PAGE and were immunoblotted for Bax.
Figure Legend Snippet: Bax translocates to mitochondria and forms high mol wt oligomers during anoikis. (a) FSK-7 cells, adherent or detached (maintained on poly-HEMA for 15 min or 4 h), were separated into soluble (S) and membrane (M) fractions before SDS-PAGE and immunoblotting with polyclonal anti-Bax (top). 4 mM CHAPS was added, and Bax was immunoprecipitated with anti-Bax 62M. Immunoprecipitates were separated by SDS-PAGE and were immunoblotted with anti-Bax 5B7 (bottom). (b) Cells expressing the OMM marker YFP-XT were detached for various times before cytospinning, and were immunostained with the activation-dependent antibody 62M. 62M reactivity is seen at discrete regions on mitochondria at all time points. Bar, 5 μm. (c) Soluble and membrane fractions from adherent cells or cells maintained on poly-HEMA for 4 h were isolated as above. CHAPS was added to a final concentration of 4 mM, and the fractions were concentrated. 5 mg protein from each fraction was loaded onto a Sephacryl S100-HR column (see Materials and methods). Separated fractions were analyzed by SDS-PAGE and immunoblotting for Bax. (d) Membrane fractions from FSK-7 cells detached for 4 h were separated by size-exclusion chromatography as above. Fractions containing high (oligo) and low (mono) mol wt Bax complexes were collected. Fractions were concentrated and separated into three equal parts. These were treated with BS3 (with or without addition of 0.1% Triton X-100). Samples were separated by SDS-PAGE and were immunoblotted for Bax. Cytosolic Bax from adherent cells (Adh) was run for comparison after identical treatment. (e) Membrane fractions isolated from adherent cells or cells detached for various times and separated by size-exclusion chromatography were analyzed by SDS-PAGE and immunoblotting for Bax. Total soluble (S) and membrane (M) fractions show Bax translocation at each time point. (f) Membrane fractions prepared from adherent cells or cells detached for 15 min, 1 h, or 4 h were spilt into three and treated with 5 mM BS3 in the presence or absence of 0.1% Triton-X100. Samples were analyzed by SDS-PAGE and were immunoblotted for Bax.

Techniques Used: SDS Page, Immunoprecipitation, Expressing, Marker, Activation Assay, Isolation, Concentration Assay, Size-exclusion Chromatography, Translocation Assay

34) Product Images from "Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura"

Article Title: Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura

Journal: PLoS ONE

doi: 10.1371/journal.pone.0160388

Characterization of the ex vivo inhibitory effect of anti-mMDTCS mAbs 13B4 and 14H7. Adamts13 +/+ mice (n = 4, per condition) were injected with 2.50 mg/kg of mAb 13B4, 14H7 or 20A10 or with a combination of mAbs 13B4 and 14H7 (1.25 mg/kg each) on day 0 (black arrow). The optimal injection dose of mAb was determined in separate experiments (data not shown). Blood was retrieved 7 days before (‘day -7’) and 1, 3, 5, 7 and 14 days post injection. (A) The influence of the different mAbs on the proteolytic activity of mADAMTS13 was determined using the FRETS-VWF73 assay. Activities were calculated based on the slope of the proteolysis reactions ( S1 Fig ). (B) Plasma mAb levels (μg/mL) were determined using ELISA. Plates were coated with recombinant mADAMST13, blocked and plasma of the respective mice was added. Bound mAbs were detected using GAM-HRP. (C) The amount of mADAMTS13 (%) in plasma was determined using ELISA. Plasma mADAMTS13 was captured using the anti-mT2-CUB2 mAb 9F2. After blocking, the respective plasma samples were added. Finally, bound mADAMTS13 was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (D) Platelet counts were measured of the respective mice samples. Error bars represent the SD (n = 4, per condition). (E) The plasma mVWF multimer pattern was determined for a new cohort of treated mice (n = 5, per condition) 7 days before (‘day -7’) and 1 and 3 days post injection of mAb(s) 20A10 or the combination of mAbs 13B4 and 14H7). Representative multimer patterns are given. Low, middle and high molecular weight (respectively LMW [1–5 bands], MMW [6–10 bands] and HMW [ > 10 bands]) multimers and UL-VWF multimers (brace) are indicated. (F) The percentage HMW multimers was calculated using the ImageJ 1.48v software.
Figure Legend Snippet: Characterization of the ex vivo inhibitory effect of anti-mMDTCS mAbs 13B4 and 14H7. Adamts13 +/+ mice (n = 4, per condition) were injected with 2.50 mg/kg of mAb 13B4, 14H7 or 20A10 or with a combination of mAbs 13B4 and 14H7 (1.25 mg/kg each) on day 0 (black arrow). The optimal injection dose of mAb was determined in separate experiments (data not shown). Blood was retrieved 7 days before (‘day -7’) and 1, 3, 5, 7 and 14 days post injection. (A) The influence of the different mAbs on the proteolytic activity of mADAMTS13 was determined using the FRETS-VWF73 assay. Activities were calculated based on the slope of the proteolysis reactions ( S1 Fig ). (B) Plasma mAb levels (μg/mL) were determined using ELISA. Plates were coated with recombinant mADAMST13, blocked and plasma of the respective mice was added. Bound mAbs were detected using GAM-HRP. (C) The amount of mADAMTS13 (%) in plasma was determined using ELISA. Plasma mADAMTS13 was captured using the anti-mT2-CUB2 mAb 9F2. After blocking, the respective plasma samples were added. Finally, bound mADAMTS13 was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (D) Platelet counts were measured of the respective mice samples. Error bars represent the SD (n = 4, per condition). (E) The plasma mVWF multimer pattern was determined for a new cohort of treated mice (n = 5, per condition) 7 days before (‘day -7’) and 1 and 3 days post injection of mAb(s) 20A10 or the combination of mAbs 13B4 and 14H7). Representative multimer patterns are given. Low, middle and high molecular weight (respectively LMW [1–5 bands], MMW [6–10 bands] and HMW [ > 10 bands]) multimers and UL-VWF multimers (brace) are indicated. (F) The percentage HMW multimers was calculated using the ImageJ 1.48v software.

Techniques Used: Ex Vivo, Mouse Assay, Injection, Activity Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Molecular Weight, Software

Development of a sensitive mADAMTS13 detection assay. (A) Binding of plasma mADAMTS13 to the anti-mADAMTS13 mAbs was tested in ELISA. The respective anti-mMDTCS (black) and anti-mT2-CUB2 (white) mAbs were coated on a 96-well microtiter plate. After blocking, plasma mADAMTS13 was added (0.1 U/mL) and was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (B) The same ELISA was performed as in A in triplicate for anti-mT2-CUB2 mAb 9F2, anti-mMDTCS mAb 14H7 and the previously reported anti-mMDTCS mAb 20A10 [ 31 ]. Binding was calculated relative to binding of 0.10 U/mL plasma mADAMTS13 to 20A10 (which was set to a value of ‘1.0’). Error bars represent the SD of the three independently performed experiments.
Figure Legend Snippet: Development of a sensitive mADAMTS13 detection assay. (A) Binding of plasma mADAMTS13 to the anti-mADAMTS13 mAbs was tested in ELISA. The respective anti-mMDTCS (black) and anti-mT2-CUB2 (white) mAbs were coated on a 96-well microtiter plate. After blocking, plasma mADAMTS13 was added (0.1 U/mL) and was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (B) The same ELISA was performed as in A in triplicate for anti-mT2-CUB2 mAb 9F2, anti-mMDTCS mAb 14H7 and the previously reported anti-mMDTCS mAb 20A10 [ 31 ]. Binding was calculated relative to binding of 0.10 U/mL plasma mADAMTS13 to 20A10 (which was set to a value of ‘1.0’). Error bars represent the SD of the three independently performed experiments.

Techniques Used: Detection Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

A subset of anti-mMDTCS mAbs inhibit plasma mADAMTS13 activity. The effect of anti-mADAMTS13 mAbs on the proteolytic activity of plasma mADAMTS13 was tested in vitro using the FRETS-VWF73 assay. (A) All anti-mADAMTS13 mAbs, both anti-mMDTCS and anti-mT2-CUB2 mAbs, were tested. For each condition, the activity of plasma mADAMTS13 was calculated using a calibration curve of 2.5%, 5%, 10% and 15% NMP. (B) Time profile of the cleavage of the FRETS-VWF73 substrate by plasma mADAMTS13, in the absence or presence of mAbs 13B4 and/or 14H7. Error bars represent the SD of at least three independently performed experiments.
Figure Legend Snippet: A subset of anti-mMDTCS mAbs inhibit plasma mADAMTS13 activity. The effect of anti-mADAMTS13 mAbs on the proteolytic activity of plasma mADAMTS13 was tested in vitro using the FRETS-VWF73 assay. (A) All anti-mADAMTS13 mAbs, both anti-mMDTCS and anti-mT2-CUB2 mAbs, were tested. For each condition, the activity of plasma mADAMTS13 was calculated using a calibration curve of 2.5%, 5%, 10% and 15% NMP. (B) Time profile of the cleavage of the FRETS-VWF73 substrate by plasma mADAMTS13, in the absence or presence of mAbs 13B4 and/or 14H7. Error bars represent the SD of at least three independently performed experiments.

Techniques Used: Activity Assay, In Vitro

Epitope mapping and epitope overview of the developed anti-mADAMTS13 mAbs. The epitope of each anti-mADAMTS13 mAb was mapped against both mMDTCS (A) and mT2-CUB2 (B). Individual anti-mADAMTS13 mAbs were coated, recombinant mMDTCS (A) or mT2-CUB2 (B) were added and binding of the respective mADAMTS13 variant was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. Black and white bars represent respectively anti-mMDTCS and anti-mT2-CUB2 mAbs. The previously reported mAb 20A10 [ 31 ] was used as a positive (A) and negative (B) control. (C) Epitope overview of the developed anti-mADAMTS13 mAbs. The previously developed mAb 20A10 [ 31 ] is marked by a dark frame.
Figure Legend Snippet: Epitope mapping and epitope overview of the developed anti-mADAMTS13 mAbs. The epitope of each anti-mADAMTS13 mAb was mapped against both mMDTCS (A) and mT2-CUB2 (B). Individual anti-mADAMTS13 mAbs were coated, recombinant mMDTCS (A) or mT2-CUB2 (B) were added and binding of the respective mADAMTS13 variant was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. Black and white bars represent respectively anti-mMDTCS and anti-mT2-CUB2 mAbs. The previously reported mAb 20A10 [ 31 ] was used as a positive (A) and negative (B) control. (C) Epitope overview of the developed anti-mADAMTS13 mAbs. The previously developed mAb 20A10 [ 31 ] is marked by a dark frame.

Techniques Used: Recombinant, Binding Assay, Variant Assay

35) Product Images from "Reinvestigation of the dysbindin subunit of BLOC-1 (biogenesis of lysosome-related organelles complex-1) as a dystrobrevin-binding protein"

Article Title: Reinvestigation of the dysbindin subunit of BLOC-1 (biogenesis of lysosome-related organelles complex-1) as a dystrobrevin-binding protein

Journal: Biochemical Journal

doi: 10.1042/BJ20051965

Co-immunoprecipitation experiments Detergent extracts prepared from mouse brain ( A ), mouse quadriceps muscle ( B ) or rat quadriceps ( C ) were subjected to immunoprecipitation using a mouse monoclonal antibody against α- and β-dystrobrevins, a rabbit polyclonal antibody to dysbindin, and comparable amounts of the indicated control antibodies. The washed immunoprecipitates were analysed by immunoblotting using monoclonal antibodies to dystrobrevin or pallidin. Small aliquots (1–4% as indicated) of the crude extracts used for immunoprecipitation were analysed in parallel. Notice in ( A , B ) that significant amounts of pallidin were specifically recovered from the sample immunoprecipitated using anti-dysbindin, and that no dystrobrevin was found in the anti-dysbindin immunoprecipitate in amounts higher than in control immunoprecipitates. Similarly, only minute amounts of α-dystrobrevin isoforms were detected in the anti-dysbindin immunoprecipitate obtained from rat quadriceps extracts, and these amounts were not higher than those of dystrobrevins non-specifically associated with immunoprecipitates obtained using irrelevant rabbit polyclonal antibodies ( C ).
Figure Legend Snippet: Co-immunoprecipitation experiments Detergent extracts prepared from mouse brain ( A ), mouse quadriceps muscle ( B ) or rat quadriceps ( C ) were subjected to immunoprecipitation using a mouse monoclonal antibody against α- and β-dystrobrevins, a rabbit polyclonal antibody to dysbindin, and comparable amounts of the indicated control antibodies. The washed immunoprecipitates were analysed by immunoblotting using monoclonal antibodies to dystrobrevin or pallidin. Small aliquots (1–4% as indicated) of the crude extracts used for immunoprecipitation were analysed in parallel. Notice in ( A , B ) that significant amounts of pallidin were specifically recovered from the sample immunoprecipitated using anti-dysbindin, and that no dystrobrevin was found in the anti-dysbindin immunoprecipitate in amounts higher than in control immunoprecipitates. Similarly, only minute amounts of α-dystrobrevin isoforms were detected in the anti-dysbindin immunoprecipitate obtained from rat quadriceps extracts, and these amounts were not higher than those of dystrobrevins non-specifically associated with immunoprecipitates obtained using irrelevant rabbit polyclonal antibodies ( C ).

Techniques Used: Immunoprecipitation

36) Product Images from "Interleukin-27 Is a Potent Inhibitor of cis HIV-1 Replication in Monocyte-Derived Dendritic Cells via a Type I Interferon-Independent Pathway"

Article Title: Interleukin-27 Is a Potent Inhibitor of cis HIV-1 Replication in Monocyte-Derived Dendritic Cells via a Type I Interferon-Independent Pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0059194

Expression levels of IL-27Rα (WSX-1) in DCs and STAT activation following IL-27 treatment. A–D: The expression level of the IL-27 receptor (WSX-1) was measured using flow cytometry on the following cells: A, Freshly isolated monocytes B, iDCs C, mDCs D, LLC. These cells were stained with either a PE-conjugated anti-WSX-1 (black) antibody or an isotype control (white). Data indicates a representative result from a single donor from three independent experiments. The numbers in the figures show percentage of positive cells, and the Mean Fluorescence Intensity (MFI) of monocytes, iDCs and mDCs were 8.75, 9.13 and 6.3, respectively. E: Western blotting was used to measure the phosphorylation status of STAT-1, -2, -3, -5, and -6 in IL-27 or untreated iDCs at a number of time points. For each phosphorylated STAT measured, the unphosphorylated protein was also measured to show that the profiles were not due to loading differences.
Figure Legend Snippet: Expression levels of IL-27Rα (WSX-1) in DCs and STAT activation following IL-27 treatment. A–D: The expression level of the IL-27 receptor (WSX-1) was measured using flow cytometry on the following cells: A, Freshly isolated monocytes B, iDCs C, mDCs D, LLC. These cells were stained with either a PE-conjugated anti-WSX-1 (black) antibody or an isotype control (white). Data indicates a representative result from a single donor from three independent experiments. The numbers in the figures show percentage of positive cells, and the Mean Fluorescence Intensity (MFI) of monocytes, iDCs and mDCs were 8.75, 9.13 and 6.3, respectively. E: Western blotting was used to measure the phosphorylation status of STAT-1, -2, -3, -5, and -6 in IL-27 or untreated iDCs at a number of time points. For each phosphorylated STAT measured, the unphosphorylated protein was also measured to show that the profiles were not due to loading differences.

Techniques Used: Expressing, Activation Assay, Flow Cytometry, Cytometry, Isolation, Staining, Fluorescence, Western Blot

The anti-HIV effects of IL-27 on DCs are not mediated by Type I IFNs. A: iDCs from three independent donors were treated with or without IL-27 for 24 hours and then RNA was extracted. An Affymetrix microarray and gene expression profile analysis were performed as described in the Materials and Methods . B: HIV-infected iDCs were pretreated with anti-IFNαR antibody (R2Ab) or isotype control antibody (Ctrl Ab) before IL-27 treatment. As an assay control, the antibody-pretreated cells were also treated with IFN-α. This graph represents one donor from three independent donors. Error bars represent SD of replicates.
Figure Legend Snippet: The anti-HIV effects of IL-27 on DCs are not mediated by Type I IFNs. A: iDCs from three independent donors were treated with or without IL-27 for 24 hours and then RNA was extracted. An Affymetrix microarray and gene expression profile analysis were performed as described in the Materials and Methods . B: HIV-infected iDCs were pretreated with anti-IFNαR antibody (R2Ab) or isotype control antibody (Ctrl Ab) before IL-27 treatment. As an assay control, the antibody-pretreated cells were also treated with IFN-α. This graph represents one donor from three independent donors. Error bars represent SD of replicates.

Techniques Used: Microarray, Expressing, Infection

HIV-1 co-receptor expression in DCs and HIV-1 infection rates in DC subsets. A: The expression level of CD4, CXCR4 and CCR5 was measured using flow cytometry in iDCs, mDCs, LLC and IL-27-treated iDCs. DC-SIGN was also measured but only in iDCs, mDCs and IL-27 treated iDCs. The red color in the plots indicates specific antibody staining whilst green represents the isotype control. B: iDCs, mDCs and LLCs were infected with either HIV-1 Ba-L or HIV-1 NL4.3 as described in the Materials and Methods . HIV-1 replication was measured using HIV-1 p24 antigen capture kit as described in the Materials and Methods . These graphs are representative data from a single donor from three independent experiments.
Figure Legend Snippet: HIV-1 co-receptor expression in DCs and HIV-1 infection rates in DC subsets. A: The expression level of CD4, CXCR4 and CCR5 was measured using flow cytometry in iDCs, mDCs, LLC and IL-27-treated iDCs. DC-SIGN was also measured but only in iDCs, mDCs and IL-27 treated iDCs. The red color in the plots indicates specific antibody staining whilst green represents the isotype control. B: iDCs, mDCs and LLCs were infected with either HIV-1 Ba-L or HIV-1 NL4.3 as described in the Materials and Methods . HIV-1 replication was measured using HIV-1 p24 antigen capture kit as described in the Materials and Methods . These graphs are representative data from a single donor from three independent experiments.

Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Staining

IL-27 inhibits HIV-1 replication in DCs in a dose-dependent manner. A: iDCs and mDCs were infected with HIV-1 Ba-L , and HIV replication was measured by the HIV-1 p24 antigen capture kit. These experiments used DCs from three donors. B: HIV-infected iDCs were cultured in the presence of different amounts of IL-27 for 14 days and HIV-1 replication was monitored by the HIV-1 p24 antigen capture kit. C: iDCs were pre-treated with either media alone (iDCs) or 100 ng/ml IL-27 for 48 hours (PreIL-27-iDCs) followed by infection with HIV-1 Ba-L . The HIV-1 infected iDCs were cultured for 14 days without IL-27 as described in the Materials and Methods and then HIV-1 replication was determined using the HIV-1 p24 antigen captures kit. The data shows a representative result from two independent donors. D: HIV-infected iDCs were cultured in the presence of 100 ng/ml of each of the IL-12 family of cytokines (IL-12, IL-23, IL-27 and IL-35) for 14 days and anti-viral effect of each cytokine was determined using the HIV p24 antigen capture kit. The results show the combined results of three independent donors and depict % of HIV-1 replication compared to untreated cells (control) and SE.
Figure Legend Snippet: IL-27 inhibits HIV-1 replication in DCs in a dose-dependent manner. A: iDCs and mDCs were infected with HIV-1 Ba-L , and HIV replication was measured by the HIV-1 p24 antigen capture kit. These experiments used DCs from three donors. B: HIV-infected iDCs were cultured in the presence of different amounts of IL-27 for 14 days and HIV-1 replication was monitored by the HIV-1 p24 antigen capture kit. C: iDCs were pre-treated with either media alone (iDCs) or 100 ng/ml IL-27 for 48 hours (PreIL-27-iDCs) followed by infection with HIV-1 Ba-L . The HIV-1 infected iDCs were cultured for 14 days without IL-27 as described in the Materials and Methods and then HIV-1 replication was determined using the HIV-1 p24 antigen captures kit. The data shows a representative result from two independent donors. D: HIV-infected iDCs were cultured in the presence of 100 ng/ml of each of the IL-12 family of cytokines (IL-12, IL-23, IL-27 and IL-35) for 14 days and anti-viral effect of each cytokine was determined using the HIV p24 antigen capture kit. The results show the combined results of three independent donors and depict % of HIV-1 replication compared to untreated cells (control) and SE.

Techniques Used: Infection, Cell Culture

Inhibition of HIV-1 replication in DCs occurs after viral entry but before completion of reverse transcription. A: iDCs were pre-treated with IL-27 or mock treated for 48 hours. The treated cells were then infected with a HIV NL4.3-Luc-VSVG virus followed by 4 days culture in the absence of IL-27 (Pre-IL-27 iDC). To compare the IL-27 effect, the mock-treated iDCs was also cultured in the presence of IL-27 (iDC+IL-27). Virus reporter activity was measured as described in the Materials and Methods B: The Late RT cDNA products were also semi quantitated using qPCR using the pseudotyped HIV infected cells. These experiments show the combined results of 4 separate donors. Error bars represent +/− SEM.
Figure Legend Snippet: Inhibition of HIV-1 replication in DCs occurs after viral entry but before completion of reverse transcription. A: iDCs were pre-treated with IL-27 or mock treated for 48 hours. The treated cells were then infected with a HIV NL4.3-Luc-VSVG virus followed by 4 days culture in the absence of IL-27 (Pre-IL-27 iDC). To compare the IL-27 effect, the mock-treated iDCs was also cultured in the presence of IL-27 (iDC+IL-27). Virus reporter activity was measured as described in the Materials and Methods B: The Late RT cDNA products were also semi quantitated using qPCR using the pseudotyped HIV infected cells. These experiments show the combined results of 4 separate donors. Error bars represent +/− SEM.

Techniques Used: Inhibition, Infection, Cell Culture, Activity Assay, Real-time Polymerase Chain Reaction

37) Product Images from "High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting"

Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

Journal: PLoS ONE

doi: 10.1371/journal.pone.0135278

Surface Plasmon Resonance sensorgrams of Abdurin variants binding to hEphrinA2. hEph2A was immobilized on CM5 chip by amine coupling with a ligand density of 500 RU. Serial dilutions of analyte in HBS-P running buffer were injected over the ligand (2.5 min association time, 5 min dissociation time) followed by surface regeneration. Sensorgrams for: (A) G7 (3.7 to 300 nM), (B) B6 (9 to 150 nM), (C) B11 (1.56 to 100 nM), and (D) B11 (red sensorgram) or shWTCH2 (black sensorgram), were analyzed with BiaEvaluation software v 3.0 and kinetic parameters or affinity at the equilibrium were evaluated.
Figure Legend Snippet: Surface Plasmon Resonance sensorgrams of Abdurin variants binding to hEphrinA2. hEph2A was immobilized on CM5 chip by amine coupling with a ligand density of 500 RU. Serial dilutions of analyte in HBS-P running buffer were injected over the ligand (2.5 min association time, 5 min dissociation time) followed by surface regeneration. Sensorgrams for: (A) G7 (3.7 to 300 nM), (B) B6 (9 to 150 nM), (C) B11 (1.56 to 100 nM), and (D) B11 (red sensorgram) or shWTCH2 (black sensorgram), were analyzed with BiaEvaluation software v 3.0 and kinetic parameters or affinity at the equilibrium were evaluated.

Techniques Used: SPR Assay, Binding Assay, Chromatin Immunoprecipitation, Injection, Software

38) Product Images from "Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication"

Article Title: Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky460

Cleavage of mismatch-containing DNA by Cgl EndoMS-WT and Cgl EndoMS-ΔCter with Cgl β-clamp. ( A ) The 5′-Cy5-labeled DNA substrates (5 nM) containing the GT base pair were incubated with the proteins. The products were analyzed by native 10% PAGE followed by laser scanning (cropped gel image). Lanes 1, positive control (20 nM TkoEndoMS, as a monomer); 2, negative control (no proteins); 3–7, 50 nM Cgl EndoMS-WT (as a monomer); 8–12, 50 nM Cgl EndoMS-ΔCter (as a monomer); 13, no EndoMS. The indicated concentrations (as a monomer) of Cgl β-clamp were added to the reactions. Representative results are shown. The band assignments are indicated on the side of the panels: s, substrates: p, cleaved products. ( B ) Quantification of the cleaved products. Independent data points from three measurements are plotted. ( C ) The physical interactions of Cgl EndoMS with Cgl β-clamp were characterized by SPR using a BIACORE J system (Biacore Inc.). Purified Cgl β-Clamp was immobilized on the sensor Chip CM5, and various concentrations (indicated on the right sides of the sensorgrams) of Cgl EndoMS-WT and Cgl EndoMS-ΔCter were loaded onto the chip for 2 min at a flow rate of 30 μl/min in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Regeneration was achieved using a 1-min injection of 0.025% SDS. Each background response was subtracted. Data were analyzed using BIAevaluation v.3 software (Biacore Inc.)
Figure Legend Snippet: Cleavage of mismatch-containing DNA by Cgl EndoMS-WT and Cgl EndoMS-ΔCter with Cgl β-clamp. ( A ) The 5′-Cy5-labeled DNA substrates (5 nM) containing the GT base pair were incubated with the proteins. The products were analyzed by native 10% PAGE followed by laser scanning (cropped gel image). Lanes 1, positive control (20 nM TkoEndoMS, as a monomer); 2, negative control (no proteins); 3–7, 50 nM Cgl EndoMS-WT (as a monomer); 8–12, 50 nM Cgl EndoMS-ΔCter (as a monomer); 13, no EndoMS. The indicated concentrations (as a monomer) of Cgl β-clamp were added to the reactions. Representative results are shown. The band assignments are indicated on the side of the panels: s, substrates: p, cleaved products. ( B ) Quantification of the cleaved products. Independent data points from three measurements are plotted. ( C ) The physical interactions of Cgl EndoMS with Cgl β-clamp were characterized by SPR using a BIACORE J system (Biacore Inc.). Purified Cgl β-Clamp was immobilized on the sensor Chip CM5, and various concentrations (indicated on the right sides of the sensorgrams) of Cgl EndoMS-WT and Cgl EndoMS-ΔCter were loaded onto the chip for 2 min at a flow rate of 30 μl/min in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Regeneration was achieved using a 1-min injection of 0.025% SDS. Each background response was subtracted. Data were analyzed using BIAevaluation v.3 software (Biacore Inc.)

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Positive Control, Negative Control, SPR Assay, Purification, Chromatin Immunoprecipitation, Flow Cytometry, Injection, Software

39) Product Images from "A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling"

Article Title: A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling

Journal: Chemistry and physics of lipids

doi: 10.1016/j.chemphyslip.2018.06.002

Two color imaging shows real time tubule fission at site of Sar1B binding. Images are shown from a single image sequence of a tubule undergoing fission. Results show a tubule prior to significant AF488-Sar1B GTP binding (A) and after binding AF488-Sar1B GTP (B-C). After the AF488-Sar1B GTP localized to the tubule base (C), the tubule disassembled into a vesicle-like structure (D), presumably due to fission. The lipid bilayer was labeled with 0.1% LRB-DOPE and excited by 561-nm laser in an epifluorescence geometry. The AF488-Sar1B GTP was excited by a 488-nm laser in TIR. (E) Fluorescence intensity vs. time graph of tubule fission event shown in (A-D). (F) Average fluorescence intensity vs. time of 5 tubule fission events. Error bars ± SEM. Scale bars 10 μm.
Figure Legend Snippet: Two color imaging shows real time tubule fission at site of Sar1B binding. Images are shown from a single image sequence of a tubule undergoing fission. Results show a tubule prior to significant AF488-Sar1B GTP binding (A) and after binding AF488-Sar1B GTP (B-C). After the AF488-Sar1B GTP localized to the tubule base (C), the tubule disassembled into a vesicle-like structure (D), presumably due to fission. The lipid bilayer was labeled with 0.1% LRB-DOPE and excited by 561-nm laser in an epifluorescence geometry. The AF488-Sar1B GTP was excited by a 488-nm laser in TIR. (E) Fluorescence intensity vs. time graph of tubule fission event shown in (A-D). (F) Average fluorescence intensity vs. time of 5 tubule fission events. Error bars ± SEM. Scale bars 10 μm.

Techniques Used: Imaging, Binding Assay, Sequencing, Labeling, Fluorescence

Sar1B membrane remodeling. The addition of Sar1B GTP to STuBs alters the morphology of lipid tubules. (A) A flexible tubule. (B) Same tubule as in (A) following addition of Sar1B GTP , exhibiting a rigid morphology. (C) A pseudo-vesiculated tubule. (D) A rigid, bifurcated tubule and a tubule exhibiting a beads-on-a-string morphology. (E) As Sar1B GTP concentration increases, the number of flexible tubules per field of view significantly decreases. (F) The prevalence of Sar1B GTP -induced structures changes as a function of concentration. Statistics by 1-way ANOVA. * indicates comparison to 0-nM Sar condition, † indicates comparison to 50-nM Sar1B condition. Adjusted P Values; *, †: P
Figure Legend Snippet: Sar1B membrane remodeling. The addition of Sar1B GTP to STuBs alters the morphology of lipid tubules. (A) A flexible tubule. (B) Same tubule as in (A) following addition of Sar1B GTP , exhibiting a rigid morphology. (C) A pseudo-vesiculated tubule. (D) A rigid, bifurcated tubule and a tubule exhibiting a beads-on-a-string morphology. (E) As Sar1B GTP concentration increases, the number of flexible tubules per field of view significantly decreases. (F) The prevalence of Sar1B GTP -induced structures changes as a function of concentration. Statistics by 1-way ANOVA. * indicates comparison to 0-nM Sar condition, † indicates comparison to 50-nM Sar1B condition. Adjusted P Values; *, †: P

Techniques Used: Concentration Assay

Fluorescently labeled Sar1B binds tubules. (A) A flexible tubule with no bound Sar1B. (B) A rigid tubule with Sar1B decorating its length. (C) A tubule exhibiting a beads-on-a-string morphology with Sar1B bound at the “beads”. Conditions: 1 nM AF488-Sar1B, 10 nM unlabeled Sar1B GTP in Buffer A + 0.5 mM MgCl 2 + 5 µM GTP. Scale bars 5 µm.
Figure Legend Snippet: Fluorescently labeled Sar1B binds tubules. (A) A flexible tubule with no bound Sar1B. (B) A rigid tubule with Sar1B decorating its length. (C) A tubule exhibiting a beads-on-a-string morphology with Sar1B bound at the “beads”. Conditions: 1 nM AF488-Sar1B, 10 nM unlabeled Sar1B GTP in Buffer A + 0.5 mM MgCl 2 + 5 µM GTP. Scale bars 5 µm.

Techniques Used: Labeling

Sar1B drives tubule fission in a GTP-dependent manner. Effect of wild-type Sar1B on tubule density in the presence of 5 µM GDP, 5 µM GTP, and 5 µM GMP-PNP. In these experiments, 50 nM Sar1B was titrated onto an SLB containing approximately 100 pre-formed tubules in each field of view sampled. *:P
Figure Legend Snippet: Sar1B drives tubule fission in a GTP-dependent manner. Effect of wild-type Sar1B on tubule density in the presence of 5 µM GDP, 5 µM GTP, and 5 µM GMP-PNP. In these experiments, 50 nM Sar1B was titrated onto an SLB containing approximately 100 pre-formed tubules in each field of view sampled. *:P

Techniques Used:

Sar1B bends membranes in a GTP-dependent manner. (A) Increases in DiD-labeled membrane curvature ( P/S ) and concentration (P + 2S ) observed after 10 nM Sar1B GTP is added to STuBs. (B-C) Graphs corresponding to the event shown in panel A. Averaged P/S (D) and P+2S (E) changes for several membrane budding events observed in the presence of Sar1B GTP (n=5). Error bars ± SEM. No similar events were observed in otherwise identical samples with Sar1B GDP or lacking protein (n ≥ 3). (G) ). Scale bars 2 µm.
Figure Legend Snippet: Sar1B bends membranes in a GTP-dependent manner. (A) Increases in DiD-labeled membrane curvature ( P/S ) and concentration (P + 2S ) observed after 10 nM Sar1B GTP is added to STuBs. (B-C) Graphs corresponding to the event shown in panel A. Averaged P/S (D) and P+2S (E) changes for several membrane budding events observed in the presence of Sar1B GTP (n=5). Error bars ± SEM. No similar events were observed in otherwise identical samples with Sar1B GDP or lacking protein (n ≥ 3). (G) ). Scale bars 2 µm.

Techniques Used: Labeling, Concentration Assay

40) Product Images from "Multiple and distinct strategies of yeast SNAREs to confer the specificity of membrane fusion"

Article Title: Multiple and distinct strategies of yeast SNAREs to confer the specificity of membrane fusion

Journal: Scientific Reports

doi: 10.1038/srep04277

The SM protein Sly1p is essential for fusogenicity of its cognate ER-Golgi SNARE complex. (a) The SM protein Sly1p specifically associates with its cognate ER-Golgi and intra-Golgi QabcR-SNARE complexes. GST-Sec22p was incubated with physiological sets of 3Q-SNAREs, which include ER-Golgi Sed5p/Bos1p/Bet1p, intra-Golgi Sed5p/Gos1p/Sft1p, endosomal Pep12p/Vti1p/Tlg1p, and vacuolar Vam3p/Vti1p/Vam7p, and Sly1p (4.6 μM final) in RB500 containing 1% Triton X-100. After GST-Sec22p was isolated by glutathione-Sepharose beads, Q-SNAREs and Sly1p bound to the beads were analyzed by SDS-PAGE and Coomassie Blue staining. Initial SNARE concentrations of all the reactions were 4 μM for each SNARE. (b) Coomassie Blue-stained gel of SNARE proteoliposomes (20 nmol total lipids in each lane) bearing the cognate ER-Golgi/intra-Golgi 3Q-SNARE sets or the Sed5p-containing mixed non-cognate 3Q-SNARE sets (Sed5p/Gos1p/Bet1p, Sed5p/Vti1p/Bet1p, Sed5p/Vti1p/Tlg1p, and Sed5p/Vti1p/Vam7p) used in (c–g). (c) Sly1p selectively binds to the Sed5p-containing 3Q-SNARE liposomes. SNARE liposomes (1 mM total lipids in final), which bore the Sed5p-containing 3Q-SNARE sets in (b), the endosomal/vacuolar 3Q-SNARE sets, or ER-Golgi/intra-Golgi R-SNARE Sec22p, were mixed with Sly1p (11 μM) in RB150, incubated (4°C, 30 min), and centrifuged (20,000 g, 4°C, 30 min). Sly1p protein co-precipitated with those SNARE liposomes was analyzed by SDS-PAGE and Coomassie Blue staining. (d–f) Sly1p strongly and exclusively activate the fusogenicity of its cognate ER-Golgi SNARE complex. Lipid mixing was assayed in RB150 containing 3.2% PEG6000, with the Sed5p-containing 3Q-SNARE liposomes (400 μM lipids final) in (b), R-SNARE liposomes (160 μM lipids final) bearing Sec22p (d), Snc2p (e), or Nyv1p (f), and Sly1p (4.5 μM final). (g) PEG-mediated tethering is required for Sly1p-dependent lipid mixing by ER-Golgi SNAREs. Lipid mixing between the cognate ER-Golgi 3Q- and R-SNARE liposomes (400 and 160 μM lipids, respectively) was assayed as in (d), in the presence of Sly1p (4.5 μM), PEG6000 (3.2%), and GST-Sec22p lacking a transmembrane domain (Sec22pΔTM, 10 μM), where indicated.
Figure Legend Snippet: The SM protein Sly1p is essential for fusogenicity of its cognate ER-Golgi SNARE complex. (a) The SM protein Sly1p specifically associates with its cognate ER-Golgi and intra-Golgi QabcR-SNARE complexes. GST-Sec22p was incubated with physiological sets of 3Q-SNAREs, which include ER-Golgi Sed5p/Bos1p/Bet1p, intra-Golgi Sed5p/Gos1p/Sft1p, endosomal Pep12p/Vti1p/Tlg1p, and vacuolar Vam3p/Vti1p/Vam7p, and Sly1p (4.6 μM final) in RB500 containing 1% Triton X-100. After GST-Sec22p was isolated by glutathione-Sepharose beads, Q-SNAREs and Sly1p bound to the beads were analyzed by SDS-PAGE and Coomassie Blue staining. Initial SNARE concentrations of all the reactions were 4 μM for each SNARE. (b) Coomassie Blue-stained gel of SNARE proteoliposomes (20 nmol total lipids in each lane) bearing the cognate ER-Golgi/intra-Golgi 3Q-SNARE sets or the Sed5p-containing mixed non-cognate 3Q-SNARE sets (Sed5p/Gos1p/Bet1p, Sed5p/Vti1p/Bet1p, Sed5p/Vti1p/Tlg1p, and Sed5p/Vti1p/Vam7p) used in (c–g). (c) Sly1p selectively binds to the Sed5p-containing 3Q-SNARE liposomes. SNARE liposomes (1 mM total lipids in final), which bore the Sed5p-containing 3Q-SNARE sets in (b), the endosomal/vacuolar 3Q-SNARE sets, or ER-Golgi/intra-Golgi R-SNARE Sec22p, were mixed with Sly1p (11 μM) in RB150, incubated (4°C, 30 min), and centrifuged (20,000 g, 4°C, 30 min). Sly1p protein co-precipitated with those SNARE liposomes was analyzed by SDS-PAGE and Coomassie Blue staining. (d–f) Sly1p strongly and exclusively activate the fusogenicity of its cognate ER-Golgi SNARE complex. Lipid mixing was assayed in RB150 containing 3.2% PEG6000, with the Sed5p-containing 3Q-SNARE liposomes (400 μM lipids final) in (b), R-SNARE liposomes (160 μM lipids final) bearing Sec22p (d), Snc2p (e), or Nyv1p (f), and Sly1p (4.5 μM final). (g) PEG-mediated tethering is required for Sly1p-dependent lipid mixing by ER-Golgi SNAREs. Lipid mixing between the cognate ER-Golgi 3Q- and R-SNARE liposomes (400 and 160 μM lipids, respectively) was assayed as in (d), in the presence of Sly1p (4.5 μM), PEG6000 (3.2%), and GST-Sec22p lacking a transmembrane domain (Sec22pΔTM, 10 μM), where indicated.

Techniques Used: Incubation, Isolation, SDS Page, Staining

ER-Golgi and intra-Golgi 3Q-SNAREs assemble into cis -QabcR-SNARE complexes exclusively with their cognate R-SNARE Sec22p, whereas endosomal and vacuolar 3Q-SNAREs associate promiscuously with non-cognate R-SNAREs. (a–h) Purified physiological sets of 3Q-SNAREs, ER-Golgi Sed5p/Bos1p/Bet1p (a, b), intra-Golgi Sed5p/Gos1p/Sft1p (c, d), endosomal Pep12p/Vti1p/Tlg1p (e, f), and vacuolar Vam3p/Vti1p/Vam7p (g, h), were mixed with GST-tagged R-SNAREs including GST-Sec22p, GST-Ykt6p, GST-Snc2p, and GST-Nyv1p where indicated, in RB500 (20 mM HEPES-NaOH, pH 7.4, 10% glycerol, 500 mM NaCl) containing 100 mM β-OG. These GST-tagged R-SNAREs were isolated by glutathione-Sepharose beads, followed by SDS-PAGE and Coomassie Blue staining, to analyze 3Q-SNAREs bound to the GST-tagged R-SNAREs. Initial SNARE concentrations of all the reactions in (a–h) were 4 μM for each SNARE protein added.
Figure Legend Snippet: ER-Golgi and intra-Golgi 3Q-SNAREs assemble into cis -QabcR-SNARE complexes exclusively with their cognate R-SNARE Sec22p, whereas endosomal and vacuolar 3Q-SNAREs associate promiscuously with non-cognate R-SNAREs. (a–h) Purified physiological sets of 3Q-SNAREs, ER-Golgi Sed5p/Bos1p/Bet1p (a, b), intra-Golgi Sed5p/Gos1p/Sft1p (c, d), endosomal Pep12p/Vti1p/Tlg1p (e, f), and vacuolar Vam3p/Vti1p/Vam7p (g, h), were mixed with GST-tagged R-SNAREs including GST-Sec22p, GST-Ykt6p, GST-Snc2p, and GST-Nyv1p where indicated, in RB500 (20 mM HEPES-NaOH, pH 7.4, 10% glycerol, 500 mM NaCl) containing 100 mM β-OG. These GST-tagged R-SNAREs were isolated by glutathione-Sepharose beads, followed by SDS-PAGE and Coomassie Blue staining, to analyze 3Q-SNAREs bound to the GST-tagged R-SNAREs. Initial SNARE concentrations of all the reactions in (a–h) were 4 μM for each SNARE protein added.

Techniques Used: Purification, Isolation, SDS Page, Staining

41) Product Images from "Chemo-enzymatic synthesis of lipid-linked GlcNAc2Man5 oligosaccharides using recombinant Alg1, Alg2 and Alg11 proteins"

Article Title: Chemo-enzymatic synthesis of lipid-linked GlcNAc2Man5 oligosaccharides using recombinant Alg1, Alg2 and Alg11 proteins

Journal: Glycobiology

doi: 10.1093/glycob/cwx045

Kinetic analysis of LLO analogs containing polyprenyl tails ( 3 ) and ( 4 ). Glycosylation experiments were performed with 20 nM TbSTT3A protein, 5 μM peptide 5CF-GSDANYTYTQ, 10 mM MnCl 2 , 150 mM NaCl, 20 mM Hepes pH 7.5, 0.035% N -dodecyl-β- d -maltopyranoside (DDM), 0.007% Cholesteryl Hemisuccinate Tris Salt (CHS) and different concentrations of synthetic LLO analogs. Data points reflect the mean of three separate measurements. Error bars indicate standard deviations. Data were fitted by nonlinear regression according to the Michaelis–Menten formula using PRISM. This figure is available in black and white in print and in color at Glycobiology online.
Figure Legend Snippet: Kinetic analysis of LLO analogs containing polyprenyl tails ( 3 ) and ( 4 ). Glycosylation experiments were performed with 20 nM TbSTT3A protein, 5 μM peptide 5CF-GSDANYTYTQ, 10 mM MnCl 2 , 150 mM NaCl, 20 mM Hepes pH 7.5, 0.035% N -dodecyl-β- d -maltopyranoside (DDM), 0.007% Cholesteryl Hemisuccinate Tris Salt (CHS) and different concentrations of synthetic LLO analogs. Data points reflect the mean of three separate measurements. Error bars indicate standard deviations. Data were fitted by nonlinear regression according to the Michaelis–Menten formula using PRISM. This figure is available in black and white in print and in color at Glycobiology online.

Techniques Used:

42) Product Images from "The electron distribution in the “activated” state of cytochrome c oxidase"

Article Title: The electron distribution in the “activated” state of cytochrome c oxidase

Journal: Scientific Reports

doi: 10.1038/s41598-018-25779-w

Absorbance difference spectra. A comparison of reduced minus oxidized difference spectra of the Cyt c O-cyt. c complex (black) and the sum of the reduced minus oxidized difference spectra of Cyt c O and cyt. c (red), respectively. Spectra of the oxidized states were recorded first and then the atmosphere in the cuvette was replaced for N 2 after which the samples were reduced with ascorbate (2 mM) and hexaammineruthenium(II) chloride (1 μM). Reduction of the samples was followed in time by recording spectra over ~2 hours until no further changes were observed. The data to the right of the axis break have been multiplied by a factor of five. The concentrations of Cyt c O and cyt. c were ~3 μM and ~5 μM, respectively, in 50 mM HEPES (pH 7.5), 0.05% DDM and 100 µM EDTA.
Figure Legend Snippet: Absorbance difference spectra. A comparison of reduced minus oxidized difference spectra of the Cyt c O-cyt. c complex (black) and the sum of the reduced minus oxidized difference spectra of Cyt c O and cyt. c (red), respectively. Spectra of the oxidized states were recorded first and then the atmosphere in the cuvette was replaced for N 2 after which the samples were reduced with ascorbate (2 mM) and hexaammineruthenium(II) chloride (1 μM). Reduction of the samples was followed in time by recording spectra over ~2 hours until no further changes were observed. The data to the right of the axis break have been multiplied by a factor of five. The concentrations of Cyt c O and cyt. c were ~3 μM and ~5 μM, respectively, in 50 mM HEPES (pH 7.5), 0.05% DDM and 100 µM EDTA.

Techniques Used:

Kinetics of absorbance changes upon reaction with O 2 . The four-electron reduced Cyt c O (black traces) or the five-electron-reduced cyt. c -Cyt c O complex (red traces) was mixed with an O 2 -saturated buffer solution. About 200 ms after mixing with the O 2 -containing buffer, at time = 0, the CO-ligand was removed by a laser flash. The four-electron complex becomes oxidized over a time scale of 8 ms (left-hand side boxes). At 445 nm ( a ) the main contribution is from redox changes at hemes a and a 3 . At 605 nm ( b ) the main contribution is from redox changes at heme a (80%) and the remaining fraction originates from changes at heme a 3 . At 830 nm ( c ) the main contribution is from Cu A where an increase in absorbance is associated with oxidation. At 550 nm ( d ) the main contribution is from redox changes at cyt. c . At 445 nm and 605 nm, the rapid change in absorbance at t = 0 is associated with CO dissociation. It is followed in time by a decrease in absorbance associated with binding of O 2 (τ ≅ 10 μs), formation of the P R state (τ ≅ 40 μs) and oxidation of the Cyt c O (τ ≅ 1.5 ms). The P R → F reaction is not seen at these wavelengths. At 830 nm two components are seen with time constants of 200 µs ( P R → F ) and 1.5 ms ( F → O ). Oxidation of cyt. c occurs over the same time scale. In the presence of cyt. c absorbance changes attributed to the Cyt c O (panels a–c) were smaller because the redox sites were re-reduced by cyt. c during the course of the reaction. Over a time scale of ~500 ms (right-hand side boxes) all redox sites become oxidized. The small increase in absorbance over this time scale in the absence of cyt. c is due to small fractional re-reduction of Cyt c O by ascorbate. Experimental conditions after mixing: 1.1 μM Cyt c O, 20 mM HEPES at pH 7.5, 0.05% DDM, 100 μM EDTA, 2 mM ascorbate, 1 μM hexa-ammine-ruthenium(II) chloride, 1 mM O 2 at ~22 °C (black trace). The red traces are averages of three traces obtained under the following conditions: [cyt. c ]/[Cyt c O] in μM ( i ) 2.1/1.6, ( ii ) 1.5/1.3, ( iii ) 1.7/1.3. The mixing ratio was 1:5 with an oxygen-saturated buffer solution (20 mM HEPES at pH 7.5). All traces have been scaled to 1 μM reacting Cyt c O based on the rapid change in absorbance at 445 nm at t = 0. A laser artifact at t = 0 has been truncated for clarity.
Figure Legend Snippet: Kinetics of absorbance changes upon reaction with O 2 . The four-electron reduced Cyt c O (black traces) or the five-electron-reduced cyt. c -Cyt c O complex (red traces) was mixed with an O 2 -saturated buffer solution. About 200 ms after mixing with the O 2 -containing buffer, at time = 0, the CO-ligand was removed by a laser flash. The four-electron complex becomes oxidized over a time scale of 8 ms (left-hand side boxes). At 445 nm ( a ) the main contribution is from redox changes at hemes a and a 3 . At 605 nm ( b ) the main contribution is from redox changes at heme a (80%) and the remaining fraction originates from changes at heme a 3 . At 830 nm ( c ) the main contribution is from Cu A where an increase in absorbance is associated with oxidation. At 550 nm ( d ) the main contribution is from redox changes at cyt. c . At 445 nm and 605 nm, the rapid change in absorbance at t = 0 is associated with CO dissociation. It is followed in time by a decrease in absorbance associated with binding of O 2 (τ ≅ 10 μs), formation of the P R state (τ ≅ 40 μs) and oxidation of the Cyt c O (τ ≅ 1.5 ms). The P R → F reaction is not seen at these wavelengths. At 830 nm two components are seen with time constants of 200 µs ( P R → F ) and 1.5 ms ( F → O ). Oxidation of cyt. c occurs over the same time scale. In the presence of cyt. c absorbance changes attributed to the Cyt c O (panels a–c) were smaller because the redox sites were re-reduced by cyt. c during the course of the reaction. Over a time scale of ~500 ms (right-hand side boxes) all redox sites become oxidized. The small increase in absorbance over this time scale in the absence of cyt. c is due to small fractional re-reduction of Cyt c O by ascorbate. Experimental conditions after mixing: 1.1 μM Cyt c O, 20 mM HEPES at pH 7.5, 0.05% DDM, 100 μM EDTA, 2 mM ascorbate, 1 μM hexa-ammine-ruthenium(II) chloride, 1 mM O 2 at ~22 °C (black trace). The red traces are averages of three traces obtained under the following conditions: [cyt. c ]/[Cyt c O] in μM ( i ) 2.1/1.6, ( ii ) 1.5/1.3, ( iii ) 1.7/1.3. The mixing ratio was 1:5 with an oxygen-saturated buffer solution (20 mM HEPES at pH 7.5). All traces have been scaled to 1 μM reacting Cyt c O based on the rapid change in absorbance at 445 nm at t = 0. A laser artifact at t = 0 has been truncated for clarity.

Techniques Used: Mass Spectrometry, Binding Assay

43) Product Images from "Involvement of Surfactant Protein D in Ebola Virus Infection Enhancement via Glycoprotein Interaction"

Article Title: Involvement of Surfactant Protein D in Ebola Virus Infection Enhancement via Glycoprotein Interaction

Journal: Viruses

doi: 10.3390/v11010015

Interaction of hSP-D with the GP of EBOV. ( A ) Binding detection via overlay assay. hSP-D was dotted onto nitrocellulose membranes and incubated with 1 µg/mL of purified HA-tagged GPΔTM. After three washes, bound GP was detected with an anti-HA tag antibody and revealed using enhanced chemiluminescence (ECL). GPΔTM (5 ng/spot) and BSA (2 µg/spot) were dotted as positive and negative controls, respectively. 1, hSP-D; 2, AP-SP-A; 3, rSP-A; 4, ficolin-2; 5, ficolin-1; 6, ficolin-3; 7, MBL; 8, BSA; 9, GPΔTM. ( B ) SPR analysis of the interaction of human collectins with immobilized GPΔTM of EBOV. Forty microliters of MBL, hSP-D, rSP-A, and AP-SP-A (2 µg/mL) were injected over 8000 RU of immobilized GPΔTM in 20 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , 0.005% surfactant P20, and pH 7.4. The specific binding signals were obtained by subtracting the background signals over a reference surface with 3600 RU of immobilized fatty-free BSA. The results shown are representative of two independent experiments.
Figure Legend Snippet: Interaction of hSP-D with the GP of EBOV. ( A ) Binding detection via overlay assay. hSP-D was dotted onto nitrocellulose membranes and incubated with 1 µg/mL of purified HA-tagged GPΔTM. After three washes, bound GP was detected with an anti-HA tag antibody and revealed using enhanced chemiluminescence (ECL). GPΔTM (5 ng/spot) and BSA (2 µg/spot) were dotted as positive and negative controls, respectively. 1, hSP-D; 2, AP-SP-A; 3, rSP-A; 4, ficolin-2; 5, ficolin-1; 6, ficolin-3; 7, MBL; 8, BSA; 9, GPΔTM. ( B ) SPR analysis of the interaction of human collectins with immobilized GPΔTM of EBOV. Forty microliters of MBL, hSP-D, rSP-A, and AP-SP-A (2 µg/mL) were injected over 8000 RU of immobilized GPΔTM in 20 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , 0.005% surfactant P20, and pH 7.4. The specific binding signals were obtained by subtracting the background signals over a reference surface with 3600 RU of immobilized fatty-free BSA. The results shown are representative of two independent experiments.

Techniques Used: Binding Assay, Overlay Assay, Incubation, Purification, SPR Assay, Injection

Characterization of the interaction of hSP-D with immobilized GPΔTM by SPR. ( A , B ) hSP-D samples (40 µL) were injected at the indicated concentrations over immobilized GPΔTM (4,700 RU, panel A) or GPΔmucΔTM (2,500 RU, panel B) in 20 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , 0.005% surfactant P20, and pH 7.4 (HBSCa-P). Fits are shown as red lines and were obtained via global fitting of the data using a 1:1 Langmuir binding model. ( C ) hSP-D (3.8 nM) was injected over GPΔTM (8000 RU) in HBSCa-P containing 5 mM mannose (Man), and 10 mM N-acetyl-glucosamine (GlcNAc) or 100 µg/mL mannan. ( D ) hNCRD and its E321K mutant (173 nM) were injected over GPΔTM (8000 RU) in HBSCa-P. (A–D) The specific binding signals shown were obtained through subtracting the background signal over a reference surface with 3600 RU of immobilized fatty acid-free BSA. The results shown are representative of two independent experiments.
Figure Legend Snippet: Characterization of the interaction of hSP-D with immobilized GPΔTM by SPR. ( A , B ) hSP-D samples (40 µL) were injected at the indicated concentrations over immobilized GPΔTM (4,700 RU, panel A) or GPΔmucΔTM (2,500 RU, panel B) in 20 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , 0.005% surfactant P20, and pH 7.4 (HBSCa-P). Fits are shown as red lines and were obtained via global fitting of the data using a 1:1 Langmuir binding model. ( C ) hSP-D (3.8 nM) was injected over GPΔTM (8000 RU) in HBSCa-P containing 5 mM mannose (Man), and 10 mM N-acetyl-glucosamine (GlcNAc) or 100 µg/mL mannan. ( D ) hNCRD and its E321K mutant (173 nM) were injected over GPΔTM (8000 RU) in HBSCa-P. (A–D) The specific binding signals shown were obtained through subtracting the background signal over a reference surface with 3600 RU of immobilized fatty acid-free BSA. The results shown are representative of two independent experiments.

Techniques Used: SPR Assay, Injection, Binding Assay, Mutagenesis

44) Product Images from "Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties"

Article Title: Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties

Journal: The Journal of Chemical Physics

doi: 10.1063/1.5019457

Normalized specific heat (Cp) endotherms and PPIase enzyme activity. (a) Normalised specific heat capacity (Cp) endotherms measured by Differential Scanning Calorimetry (DSC) for WTCyp40, K227ACyp40, K308ACyp40, and K227A/K308ACyp40. DSC endotherms for the melting of WTCyp40 (black), K227ACyp40 (magenta), K308ACyp40 (blue), and K227A/K308ACyp40 (gray) show mutating lysine 227 and lysine 308 reduces the enthalpy of unfolding of Cyp40. The calorimetric enthalpy, ΔH, corresponds to the area under the curve. Contributions of site point mutants to the enthalpy of unfolding are additive (see Table II for values). Samples were scanned from 5 to 85 °C at a scan rate of 60 °C/h after a 5-min pre-scan equilibration in 50 mM HEPES, pH8, 150 mM sodium chloride, and 1 mM DTT. Data were baseline corrected by subtracting a buffer scan collected under the same conditions as the protein samples and normalized with respect to protein concentration. Thermodynamic parameters were determined using the software provided by the manufacturer (Origin, 7.0). All data were collected in triplicate. (b) Mutation of lysine 227 and lysine 308 enhance PPIase enzyme activity. Comparison of the biochemical activity of WTCyp40, K227ACyp40, K308ACyp40, and K227A/K308ACyp40. Cyp40 catalyses the cis-trans isomerisation of the tetrapeptide peptide substrate s-ALPF- p -nitroaniline. Mutating lysine 227 and lysine 308 enhances the activity of Cyp40. The final solution contained 20 nM Cyp40; 0.6 mg ml −1 α-chymotrypsin; 120 μ M s-ALPF- p -nitroaniline; 50 mM HEPES, pH8; 100 mM sodium chloride 14 μ M lithium chloride; 1 mM DTT; 0.5 mM EDTA; 3% 2,2,2-trifluoroethanol (v/v). Enzyme catalysed turnover has been corrected for the thermal turnover of the substrate. Data represent the mean of 6 replicates with the standard error.
Figure Legend Snippet: Normalized specific heat (Cp) endotherms and PPIase enzyme activity. (a) Normalised specific heat capacity (Cp) endotherms measured by Differential Scanning Calorimetry (DSC) for WTCyp40, K227ACyp40, K308ACyp40, and K227A/K308ACyp40. DSC endotherms for the melting of WTCyp40 (black), K227ACyp40 (magenta), K308ACyp40 (blue), and K227A/K308ACyp40 (gray) show mutating lysine 227 and lysine 308 reduces the enthalpy of unfolding of Cyp40. The calorimetric enthalpy, ΔH, corresponds to the area under the curve. Contributions of site point mutants to the enthalpy of unfolding are additive (see Table II for values). Samples were scanned from 5 to 85 °C at a scan rate of 60 °C/h after a 5-min pre-scan equilibration in 50 mM HEPES, pH8, 150 mM sodium chloride, and 1 mM DTT. Data were baseline corrected by subtracting a buffer scan collected under the same conditions as the protein samples and normalized with respect to protein concentration. Thermodynamic parameters were determined using the software provided by the manufacturer (Origin, 7.0). All data were collected in triplicate. (b) Mutation of lysine 227 and lysine 308 enhance PPIase enzyme activity. Comparison of the biochemical activity of WTCyp40, K227ACyp40, K308ACyp40, and K227A/K308ACyp40. Cyp40 catalyses the cis-trans isomerisation of the tetrapeptide peptide substrate s-ALPF- p -nitroaniline. Mutating lysine 227 and lysine 308 enhances the activity of Cyp40. The final solution contained 20 nM Cyp40; 0.6 mg ml −1 α-chymotrypsin; 120 μ M s-ALPF- p -nitroaniline; 50 mM HEPES, pH8; 100 mM sodium chloride 14 μ M lithium chloride; 1 mM DTT; 0.5 mM EDTA; 3% 2,2,2-trifluoroethanol (v/v). Enzyme catalysed turnover has been corrected for the thermal turnover of the substrate. Data represent the mean of 6 replicates with the standard error.

Techniques Used: Activity Assay, Protein Concentration, Software, Mutagenesis

45) Product Images from "Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP"

Article Title: Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP

Journal: Acta Crystallographica. Section F, Structural Biology Communications

doi: 10.1107/S2053230X13034705

Melting curve. A temperature melting curve of AtzF was performed in triplicate, using differential scanning fluorimetry with the dye SYPRO Orange. The red curves are the AtzF protein (in triplicate) in the buffer used for crystallization (50 m M HEPES, 100 m M NaCl pH 7.5). The blue curves are a 0.1 mg ml −1 lysozyme control.
Figure Legend Snippet: Melting curve. A temperature melting curve of AtzF was performed in triplicate, using differential scanning fluorimetry with the dye SYPRO Orange. The red curves are the AtzF protein (in triplicate) in the buffer used for crystallization (50 m M HEPES, 100 m M NaCl pH 7.5). The blue curves are a 0.1 mg ml −1 lysozyme control.

Techniques Used: Crystallization Assay

46) Product Images from "Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly"

Article Title: Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.2932

Binding of M. jannaschii FlaH to immobilized ATP. 10 μg of FlaH were incubated with 20 μL of ATP‐agarose in 20 m M HEPES, pH 8.0, 100 m M NaCl, 5 m M MgCl 2 for 2 h at 30°C. Samples were analyzed by SDS‐PAGE. 1, loaded protein; 2, supernatant after incubation; 3, ATP elution; 4, elution with SDS‐PAAG loading buffer. Agarose was used as a blank control.
Figure Legend Snippet: Binding of M. jannaschii FlaH to immobilized ATP. 10 μg of FlaH were incubated with 20 μL of ATP‐agarose in 20 m M HEPES, pH 8.0, 100 m M NaCl, 5 m M MgCl 2 for 2 h at 30°C. Samples were analyzed by SDS‐PAGE. 1, loaded protein; 2, supernatant after incubation; 3, ATP elution; 4, elution with SDS‐PAAG loading buffer. Agarose was used as a blank control.

Techniques Used: Binding Assay, Incubation, SDS Page

47) Product Images from "FMN binding site of yeast NADPH-cytochrome P450 reductase exposed at the surface is highly specific"

Article Title: FMN binding site of yeast NADPH-cytochrome P450 reductase exposed at the surface is highly specific

Journal: ACS chemical biology

doi: 10.1021/cb100055v

Sensograms of yCPR immobilization on Biacore chips A . yCPR immobilization on Ni-NTA chip. Channel 1: activated Ni-NTA surface, channel 2: non-activated NTA surface. HBS, NTA chip running buffer; yCPR, 100 nM yCPR in HBS running buffer; EDTA, NTA chip regeneration solution. B . yCPR immobilization on CM5 chip. EDC/NHS, CM5 surface activation solution, HBS-EP, CM5 chip running buffer; yCPR, 0.65 μM yCPR in 10 mM acetate buffer, pH 5.5; ethanolamine, 1 M ethanolamine, pH 8.5. Im = quantity of immobilized yCPR in RU (1 RU = 1 picogram of protein).
Figure Legend Snippet: Sensograms of yCPR immobilization on Biacore chips A . yCPR immobilization on Ni-NTA chip. Channel 1: activated Ni-NTA surface, channel 2: non-activated NTA surface. HBS, NTA chip running buffer; yCPR, 100 nM yCPR in HBS running buffer; EDTA, NTA chip regeneration solution. B . yCPR immobilization on CM5 chip. EDC/NHS, CM5 surface activation solution, HBS-EP, CM5 chip running buffer; yCPR, 0.65 μM yCPR in 10 mM acetate buffer, pH 5.5; ethanolamine, 1 M ethanolamine, pH 8.5. Im = quantity of immobilized yCPR in RU (1 RU = 1 picogram of protein).

Techniques Used: Chromatin Immunoprecipitation, Activation Assay

48) Product Images from "MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties"

Article Title: MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.26.3.843-851.2006

MBD2 recruits PRMT5 to chromatin. (A) Schematic representation of the primer sets used in the ChIP experiments. Exons are indicated with black rectangles. CpG islands are indicated in gray. Primer pairs are indicated with arrows. (B) ChIP analysis of MBD2, MBD3, PRMT5, and MTA2 in MCF7 cells. Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three independent chromatin isolations. (C) ChIP analysis of MCF7 cells treated with 5-azacytidine. Immunoprecipitations were performed with antibodies against MBD2, MBD3, MTA2, PRMT5, and anti-dimethyl-histone H4 (Arg3). Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three independent chromatin isolations.
Figure Legend Snippet: MBD2 recruits PRMT5 to chromatin. (A) Schematic representation of the primer sets used in the ChIP experiments. Exons are indicated with black rectangles. CpG islands are indicated in gray. Primer pairs are indicated with arrows. (B) ChIP analysis of MBD2, MBD3, PRMT5, and MTA2 in MCF7 cells. Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three independent chromatin isolations. (C) ChIP analysis of MCF7 cells treated with 5-azacytidine. Immunoprecipitations were performed with antibodies against MBD2, MBD3, MTA2, PRMT5, and anti-dimethyl-histone H4 (Arg3). Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three independent chromatin isolations.

Techniques Used: Chromatin Immunoprecipitation

PRMT5 interacts with and methylates the N-terminal RG-rich repeat of MBD2. (A) Purified TAP-MBD2 and TAP-MBD3 complexes were analyzed by Western blotting using a PRMT5 antibody. (B) Sequence of MBD2 with the RG repeats being underlined. (C) In vitro methylation of purified MBD2 complex upon incubation with S -[ 14 C]adenosylmethionine. Methylated protein is indicated with an arrow. Free label is indicated with an asterisk. MBD2 complex was purified using IgG beads. The left panel depicts a Western blot analysis of the purified MBD2 complex or a 293 control purification using anti-ProtA-horseradish peroxidase antibody. The arrow shows TAP-tagged MBD2. (D) (Left panels) In vitro methylation of recombinant GST-RG(n) and GST-MBD2 in the presence of S -[ 14 C]adenosylmethionine and a purified PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). GST-PAH2 was used as a negative control. (Right panels) In vitro methylation of recombinant GST-RG(n), GST-MBD2 lacking the RG stretch, and GST-MBD3 in the presence of S -[ 14 C]adenosylmethionine and a purified PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). Free label is indicated with an asterisk. Loading controls for GST-PAH2, GST-RG(n), GST-MBD2, GST MBD2 lacking the RG stretch, and GST-MBD3 are shown in the bottom panels. (E) Silver-stained gel of purified N-terminally truncated MBD2 complex from HEK 293 cells. The table shows the FT-MS/MS analysis with all identified proteins and their respective peptide numbers and percent sequence coverage.
Figure Legend Snippet: PRMT5 interacts with and methylates the N-terminal RG-rich repeat of MBD2. (A) Purified TAP-MBD2 and TAP-MBD3 complexes were analyzed by Western blotting using a PRMT5 antibody. (B) Sequence of MBD2 with the RG repeats being underlined. (C) In vitro methylation of purified MBD2 complex upon incubation with S -[ 14 C]adenosylmethionine. Methylated protein is indicated with an arrow. Free label is indicated with an asterisk. MBD2 complex was purified using IgG beads. The left panel depicts a Western blot analysis of the purified MBD2 complex or a 293 control purification using anti-ProtA-horseradish peroxidase antibody. The arrow shows TAP-tagged MBD2. (D) (Left panels) In vitro methylation of recombinant GST-RG(n) and GST-MBD2 in the presence of S -[ 14 C]adenosylmethionine and a purified PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). GST-PAH2 was used as a negative control. (Right panels) In vitro methylation of recombinant GST-RG(n), GST-MBD2 lacking the RG stretch, and GST-MBD3 in the presence of S -[ 14 C]adenosylmethionine and a purified PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). Free label is indicated with an asterisk. Loading controls for GST-PAH2, GST-RG(n), GST-MBD2, GST MBD2 lacking the RG stretch, and GST-MBD3 are shown in the bottom panels. (E) Silver-stained gel of purified N-terminally truncated MBD2 complex from HEK 293 cells. The table shows the FT-MS/MS analysis with all identified proteins and their respective peptide numbers and percent sequence coverage.

Techniques Used: Purification, Western Blot, Sequencing, In Vitro, Methylation, Incubation, Recombinant, Negative Control, Staining, Mass Spectrometry

49) Product Images from "Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent"

Article Title: Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent

Journal: PLoS ONE

doi: 10.1371/journal.pone.0174024

Surface plasmon resonance (SPR) analysis of temporin binding to negatively charged DMPC/DMPG 3:1 (mol:mol) LUVs. A, binding of temporins directly to the L1 sensor chip surface. SHa and [K 3 ]SHa injected at a concentration of 5 μ M (20 μ l during 1 min) interact with the carboxymethylated dextran containing covalently attached alkyl chains, as indicated by the significant amount of temporin non-specific binding (SHa: 197 RU, [K 3 ]SHa: 240 RU) remaining on the sensor chip surface after the end of peptide injection. B and C, binding of SHa (B) and [K 3 ]SHa (C) after injection of BSA. In contrast, no peptide interaction was observed after binding of 0.2 mg/ml BSA (15 μ l injected during 3 min) to the sensor chip surface followed by injection of SHa or [K 3 ]SHa (5 μ M). D, complete SPR cycle used for the binding of temporins. In the example, 0.2 mg/ml BSA was first injected (15 μ l during 3 min) on the L1 surface to prevent non-specific binding of temporins and was followed by an injection (2 μ l during 2 min) of 0.2 mg/ml DMPC/DMPG LUVs and then of peptide (300 nM of SHa in the example; 20 μ l during 1 min). Complete regeneration of the surface was obtained using 40 mM of the detergent n -octyl-β-D-glucopyranoside (OG) (30 μ l injected during 1 min). E and F, determination of the binding affinity of temporins SHa (E) and [K 3 ]SHa (F). Peptides diluted in HBS-N buffer were tested at different concentrations (0 to 300 nM) for their binding to DMPC/DMPG LUVs. The baseline corresponds to HBS-N alone. The following K D values were calculated by BIAevaluation software analysis: K D (SHa) = 1.3 ± 0.4 x 10 −7 M, χ 2 = 3.7 ± 1.3 (n = 3); K D ([K 3 ]SHa) = 3.1 ± 0.7 x 10 −8 M, χ 2 = 3.2 ± 0.7 (n = 3). Chi 2 (χ 2 ) values below 10 indicate a good fit of the Langmuir (1:1) binding model. G and H, selective SPR binding of temporins SHa (G) and [K 3 ]SHa (H) toward anionic model membranes. Negatively charged DMPG or zwitterionic DMPC LUVs were injected onto the L1 sensor chip precoated with BSA (0.2 mg/ml). Temporins (500 nM) were then injected, and binding to the DMPG (solid line) and DMPC (dashed line) LUVs was monitored. RU: resonance units; SI: start of injection; EI: end of injection. The curves correspond to a single experiment representative of three different experiments.
Figure Legend Snippet: Surface plasmon resonance (SPR) analysis of temporin binding to negatively charged DMPC/DMPG 3:1 (mol:mol) LUVs. A, binding of temporins directly to the L1 sensor chip surface. SHa and [K 3 ]SHa injected at a concentration of 5 μ M (20 μ l during 1 min) interact with the carboxymethylated dextran containing covalently attached alkyl chains, as indicated by the significant amount of temporin non-specific binding (SHa: 197 RU, [K 3 ]SHa: 240 RU) remaining on the sensor chip surface after the end of peptide injection. B and C, binding of SHa (B) and [K 3 ]SHa (C) after injection of BSA. In contrast, no peptide interaction was observed after binding of 0.2 mg/ml BSA (15 μ l injected during 3 min) to the sensor chip surface followed by injection of SHa or [K 3 ]SHa (5 μ M). D, complete SPR cycle used for the binding of temporins. In the example, 0.2 mg/ml BSA was first injected (15 μ l during 3 min) on the L1 surface to prevent non-specific binding of temporins and was followed by an injection (2 μ l during 2 min) of 0.2 mg/ml DMPC/DMPG LUVs and then of peptide (300 nM of SHa in the example; 20 μ l during 1 min). Complete regeneration of the surface was obtained using 40 mM of the detergent n -octyl-β-D-glucopyranoside (OG) (30 μ l injected during 1 min). E and F, determination of the binding affinity of temporins SHa (E) and [K 3 ]SHa (F). Peptides diluted in HBS-N buffer were tested at different concentrations (0 to 300 nM) for their binding to DMPC/DMPG LUVs. The baseline corresponds to HBS-N alone. The following K D values were calculated by BIAevaluation software analysis: K D (SHa) = 1.3 ± 0.4 x 10 −7 M, χ 2 = 3.7 ± 1.3 (n = 3); K D ([K 3 ]SHa) = 3.1 ± 0.7 x 10 −8 M, χ 2 = 3.2 ± 0.7 (n = 3). Chi 2 (χ 2 ) values below 10 indicate a good fit of the Langmuir (1:1) binding model. G and H, selective SPR binding of temporins SHa (G) and [K 3 ]SHa (H) toward anionic model membranes. Negatively charged DMPG or zwitterionic DMPC LUVs were injected onto the L1 sensor chip precoated with BSA (0.2 mg/ml). Temporins (500 nM) were then injected, and binding to the DMPG (solid line) and DMPC (dashed line) LUVs was monitored. RU: resonance units; SI: start of injection; EI: end of injection. The curves correspond to a single experiment representative of three different experiments.

Techniques Used: SPR Assay, Binding Assay, Chromatin Immunoprecipitation, Injection, Concentration Assay, Software

50) Product Images from "Analysis of 13C and 14C labeling in pyruvate and lactate in tumor and blood of lymphoma‐bearing mice injected with 13C‐ and 14C‐labeled pyruvate, et al. Analysis of 13C and 14C labeling in pyruvate and lactate in tumor and blood of lymphoma‐bearing mice injected with 13C‐ and 14C‐labeled pyruvate"

Article Title: Analysis of 13C and 14C labeling in pyruvate and lactate in tumor and blood of lymphoma‐bearing mice injected with 13C‐ and 14C‐labeled pyruvate, et al. Analysis of 13C and 14C labeling in pyruvate and lactate in tumor and blood of lymphoma‐bearing mice injected with 13C‐ and 14C‐labeled pyruvate

Journal: Nmr in Biomedicine

doi: 10.1002/nbm.3901

Images of pyruvate and lactate acquired 15 s after injection of hyperpolarized [1‐ 13 C]pyruvate in a tumor‐bearing mouse (A). The grayscale image is an anatomical  1 H image of tissue water. The blood vessel (yellow), tumor (red) and bowel (blue) regions are outlined. The false‐color images show the intensities of the pyruvate (172.9 ppm) and lactate (185.1 ppm) signals normalized to the maximum pyruvate signal in the slice. A lactate image from the same slice, multiplied by a factor of five, is also shown. Summed spectra from the blood vessel, bowel and tumor regions are shown below the corresponding images (B). The  y ‐scale for the bowel and tumor spectra has been multiplied by a factor of five to aid visualization. The broken line in the tumor spectrum shows the blood vessel spectrum, which has been scaled to take account of the fact that the blood volume is ~2% of the tumor volume
Figure Legend Snippet: Images of pyruvate and lactate acquired 15 s after injection of hyperpolarized [1‐ 13 C]pyruvate in a tumor‐bearing mouse (A). The grayscale image is an anatomical 1 H image of tissue water. The blood vessel (yellow), tumor (red) and bowel (blue) regions are outlined. The false‐color images show the intensities of the pyruvate (172.9 ppm) and lactate (185.1 ppm) signals normalized to the maximum pyruvate signal in the slice. A lactate image from the same slice, multiplied by a factor of five, is also shown. Summed spectra from the blood vessel, bowel and tumor regions are shown below the corresponding images (B). The y ‐scale for the bowel and tumor spectra has been multiplied by a factor of five to aid visualization. The broken line in the tumor spectrum shows the blood vessel spectrum, which has been scaled to take account of the fact that the blood volume is ~2% of the tumor volume

Techniques Used: Injection

Effect of injection of a gadolinium chelate (Prohance) on the tumor lactate signal intensity. The contrast agent was injected ~35 s after injection of hyperpolarized [1‐ 13 C]pyruvate at  t  = 0. The full line shows a fit to Equation   (1) . The broken line shows the expected lactate signal intensity if the gadolinium (Gd) chelate had not been injected. 4‐CIN, α‐cyano‐4‐hydroxycinnamate
Figure Legend Snippet: Effect of injection of a gadolinium chelate (Prohance) on the tumor lactate signal intensity. The contrast agent was injected ~35 s after injection of hyperpolarized [1‐ 13 C]pyruvate at t = 0. The full line shows a fit to Equation  (1) . The broken line shows the expected lactate signal intensity if the gadolinium (Gd) chelate had not been injected. 4‐CIN, α‐cyano‐4‐hydroxycinnamate

Techniques Used: Injection

51) Product Images from "Arc is a flexible modular protein capable of reversible self-oligomerization"

Article Title: Arc is a flexible modular protein capable of reversible self-oligomerization

Journal: Biochemical Journal

doi: 10.1042/BJ20141446

Interaction of PS1 with hArc ( A ) SPR sensorgrams showing binding of the PS1 peptide (unbroken lines) to hArc with increasing peptide concentrations (up to 400 μM) at 25°C in HBS-EP, pH 7.4, buffer. A K d value of 42±8 μM was obtained from the analysis of the sensorgrams by fitting a 1:1 Langmuir binding model. Peptide A (dashed line showing result at 400 μM) was used as a negative control and showed very weak binding. ( B ) Far-UV CD spectra of hArc (green), PS1 (orange) and hArc together with PS1 (red). The concentrations were 4 μM for hArc and 10 μM for PS1 and samples were prepared in 10 mM potassium phosphate, pH 7.4. The CD spectra were taken at 20°C and are buffer-subtracted. The blue dashed line represents the arithmetic addition of the CD spectra for hArc and PS1 alone.
Figure Legend Snippet: Interaction of PS1 with hArc ( A ) SPR sensorgrams showing binding of the PS1 peptide (unbroken lines) to hArc with increasing peptide concentrations (up to 400 μM) at 25°C in HBS-EP, pH 7.4, buffer. A K d value of 42±8 μM was obtained from the analysis of the sensorgrams by fitting a 1:1 Langmuir binding model. Peptide A (dashed line showing result at 400 μM) was used as a negative control and showed very weak binding. ( B ) Far-UV CD spectra of hArc (green), PS1 (orange) and hArc together with PS1 (red). The concentrations were 4 μM for hArc and 10 μM for PS1 and samples were prepared in 10 mM potassium phosphate, pH 7.4. The CD spectra were taken at 20°C and are buffer-subtracted. The blue dashed line represents the arithmetic addition of the CD spectra for hArc and PS1 alone.

Techniques Used: SPR Assay, Binding Assay, Negative Control

52) Product Images from "VIPP1 Involved in Chloroplast Membrane Integrity Has GTPase Activity in Vitro 1VIPP1 Involved in Chloroplast Membrane Integrity Has GTPase Activity in Vitro 1 [OPEN]"

Article Title: VIPP1 Involved in Chloroplast Membrane Integrity Has GTPase Activity in Vitro 1VIPP1 Involved in Chloroplast Membrane Integrity Has GTPase Activity in Vitro 1 [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.18.00145

Preparation of recombinant VIPP1-His proteins. A, Expression of recombinant VIPP1-His proteins in E. coli cells, which was confirmed by SDS-PAGE and either Coomassie Brilliant Blue (CBB) staining or immunoblot analyses. B, Composition of protein solutions used for this study. The final protein preparation (0.2 µg protein lane −1 ) was subjected to SDS-PAGE and Coomassie Brilliant Blue staining to confirm most VIPP1-His proteins.
Figure Legend Snippet: Preparation of recombinant VIPP1-His proteins. A, Expression of recombinant VIPP1-His proteins in E. coli cells, which was confirmed by SDS-PAGE and either Coomassie Brilliant Blue (CBB) staining or immunoblot analyses. B, Composition of protein solutions used for this study. The final protein preparation (0.2 µg protein lane −1 ) was subjected to SDS-PAGE and Coomassie Brilliant Blue staining to confirm most VIPP1-His proteins.

Techniques Used: Recombinant, Expressing, SDS Page, Staining

Schematic model for the structure of recombinant VIPP1 proteins used in this study. A, Schematic illustration of Arabidopsis VIPP1 secondary structure, indicating the total length and different truncated VIPP1 proteins. H denotes the predicted α-helix structure of VIPP1. The numbers indicate the positions of first (left) and last (right) amino acids of VIPP1 proteins of different lengths. WT, Wild type. B, Point mutations of amino acids in the first α-helix at the N terminus of VIPP1. The amino acids are positioned with the corresponding number, in which mutated residues are red. The substituted amino acids are listed below and are indicated with arrows.
Figure Legend Snippet: Schematic model for the structure of recombinant VIPP1 proteins used in this study. A, Schematic illustration of Arabidopsis VIPP1 secondary structure, indicating the total length and different truncated VIPP1 proteins. H denotes the predicted α-helix structure of VIPP1. The numbers indicate the positions of first (left) and last (right) amino acids of VIPP1 proteins of different lengths. WT, Wild type. B, Point mutations of amino acids in the first α-helix at the N terminus of VIPP1. The amino acids are positioned with the corresponding number, in which mutated residues are red. The substituted amino acids are listed below and are indicated with arrows.

Techniques Used: Recombinant

53) Product Images from "Stability, structural and functional properties of a monomeric, calcium–loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis"

Article Title: Stability, structural and functional properties of a monomeric, calcium–loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis

Journal: Scientific Reports

doi: 10.1038/srep42065

Identification of proteolytic sites in hCyaAm in the presence of 0.5 or 2 mM calcium. A batch of hCyaAm at a final concentration of 0.8 μM in buffer A complemented with 0.5 or 2 mM CaCl 2 was incubated at 20 °C with trypsin at a final concentration of 20 nM. A sample of hCyaAm without trypsin was included as negative control. Trypsin reaction was stopped by adding AEBSF at final concentration of 200 μM and by plunging the samples into liquid nitrogen. Black stars (★) represent the proteolytic sites identified by mass spectrometry in the presence of 0.5 mM CaCl 2 (upper panel) or in the presence of 2 mM CaCl 2 (middle panel). The fraction of proteolysis sites over the number of amino acids in each region is 7, 11, 1.5, 5 and 3% in ACD, TR, HR, AR and RD, respectively. The same experiment has been performed on the isolated RD domain (residues 1001–1706). Black stars (★) represent proteolytic sites identified in 0.5 mM calcium, violet stars ( ) correspond to cuts identified in 2 mM calcium and open stars (☆) are proteolytic sites identified in both, 0.5 and 2 mM calcium. All proteolytic sites are listed in Tables S1 to S4 . The orange boxes below the CyaA sequences correspond to the cleavage sites identified in CyaA and absent in RD while orange boxes below RD sequence correspond to proteolytic sites observed in RD only. These latter proteolytic sites, labeled in red on the SAXS-derived model of holo-RD (34), highlight the regions stabilized in the full-length toxin by the presence of other domains. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Two independent preparations of hCyaAm were used for this experiment.
Figure Legend Snippet: Identification of proteolytic sites in hCyaAm in the presence of 0.5 or 2 mM calcium. A batch of hCyaAm at a final concentration of 0.8 μM in buffer A complemented with 0.5 or 2 mM CaCl 2 was incubated at 20 °C with trypsin at a final concentration of 20 nM. A sample of hCyaAm without trypsin was included as negative control. Trypsin reaction was stopped by adding AEBSF at final concentration of 200 μM and by plunging the samples into liquid nitrogen. Black stars (★) represent the proteolytic sites identified by mass spectrometry in the presence of 0.5 mM CaCl 2 (upper panel) or in the presence of 2 mM CaCl 2 (middle panel). The fraction of proteolysis sites over the number of amino acids in each region is 7, 11, 1.5, 5 and 3% in ACD, TR, HR, AR and RD, respectively. The same experiment has been performed on the isolated RD domain (residues 1001–1706). Black stars (★) represent proteolytic sites identified in 0.5 mM calcium, violet stars ( ) correspond to cuts identified in 2 mM calcium and open stars (☆) are proteolytic sites identified in both, 0.5 and 2 mM calcium. All proteolytic sites are listed in Tables S1 to S4 . The orange boxes below the CyaA sequences correspond to the cleavage sites identified in CyaA and absent in RD while orange boxes below RD sequence correspond to proteolytic sites observed in RD only. These latter proteolytic sites, labeled in red on the SAXS-derived model of holo-RD (34), highlight the regions stabilized in the full-length toxin by the presence of other domains. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Two independent preparations of hCyaAm were used for this experiment.

Techniques Used: Concentration Assay, Incubation, Negative Control, Mass Spectrometry, Isolation, Sequencing, Labeling, Derivative Assay

SAXS study of hCyaAm. Panel (A) UV elution profile of CyaA from the size exclusion chromatography column Bio SEC-3 equilibrated in buffer A complemented with 4 mM CaCl 2 in-line with the SAXS measuring cell. Panel (B) distance distribution function P(r) derived from hCyaAm scattering pattern scaled to I(0). The dashed green line corresponds to the P(r) curve obtained by injecting the sample into the size exclusion chromatography column equilibrated with buffer A complemented with 2 mM EDTA. Panel (C) dimensionless Kratky plot of hCyaAm scattering pattern (red line) and PolX scattering pattern (black line, PolX is a compact, fully structured, globular protein 80 ). Panel (D) Two views of the most typical DAMMIN model of CyaA. Top/bottom views are rotated by 90°. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Two independent preparations of hCyaAm were used for this experiment.
Figure Legend Snippet: SAXS study of hCyaAm. Panel (A) UV elution profile of CyaA from the size exclusion chromatography column Bio SEC-3 equilibrated in buffer A complemented with 4 mM CaCl 2 in-line with the SAXS measuring cell. Panel (B) distance distribution function P(r) derived from hCyaAm scattering pattern scaled to I(0). The dashed green line corresponds to the P(r) curve obtained by injecting the sample into the size exclusion chromatography column equilibrated with buffer A complemented with 2 mM EDTA. Panel (C) dimensionless Kratky plot of hCyaAm scattering pattern (red line) and PolX scattering pattern (black line, PolX is a compact, fully structured, globular protein 80 ). Panel (D) Two views of the most typical DAMMIN model of CyaA. Top/bottom views are rotated by 90°. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Two independent preparations of hCyaAm were used for this experiment.

Techniques Used: Size-exclusion Chromatography, Derivative Assay

LUV permeabilization by various CyaA species. Panel (A) Amplitudes of ANTS fluorescence changes induced by monomeric holo-CyaA (hCyaAm, red circles), multimeric CyaA (M-CyaA, blue squares) and CyaA diluted from the urea stock solution (U-CyaA, green diamonds). A final concentration of 50 nM of CyaA was added to a LUV solution of 500 μM of lipids. The lipids composition of the LUV was POPC:POPG:Chol in a ratio of 3:1:1. Panel (B) Permeabilization of LUV by the different CyaA species (same color code as in panel A). Panel (C) Permeabilization of LUV (pre-incubated in buffer A) by different CyaA species pre-incubated in buffer A complemented with 2 mM CaCl 2 . Panel (D) Permeabilization of LUV (pre-incubated in buffer A and complemented with 3 mM EDTA) by the different CyaA species pre-incubated in buffer A. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Standard deviation values in panel (A) ±30 fluorescence intensity units. Four independent preparations of CyaA were used for this set of experiments.
Figure Legend Snippet: LUV permeabilization by various CyaA species. Panel (A) Amplitudes of ANTS fluorescence changes induced by monomeric holo-CyaA (hCyaAm, red circles), multimeric CyaA (M-CyaA, blue squares) and CyaA diluted from the urea stock solution (U-CyaA, green diamonds). A final concentration of 50 nM of CyaA was added to a LUV solution of 500 μM of lipids. The lipids composition of the LUV was POPC:POPG:Chol in a ratio of 3:1:1. Panel (B) Permeabilization of LUV by the different CyaA species (same color code as in panel A). Panel (C) Permeabilization of LUV (pre-incubated in buffer A) by different CyaA species pre-incubated in buffer A complemented with 2 mM CaCl 2 . Panel (D) Permeabilization of LUV (pre-incubated in buffer A and complemented with 3 mM EDTA) by the different CyaA species pre-incubated in buffer A. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Standard deviation values in panel (A) ±30 fluorescence intensity units. Four independent preparations of CyaA were used for this set of experiments.

Techniques Used: Fluorescence, Concentration Assay, Incubation, Standard Deviation

Thermal stability of hCyaAm followed by tryptophan fluorescence. Panel (A) Temperature-induced unfolding of hCyaAm at 50 nM followed by intrinsic fluorescence of tryptophan using the ratio of fluorescence emission intensities at 320 nm and 360 nm (rFI 320/360) as described in Materials and Methods. Panel (B) Effect of ionic strength and the presence of the molecular crowding agent Ficoll 100 g/L on the stability of hCyaAm as a function of calcium concentration ( i.e. , 0, 0.2, 0.5, 1, 2 and 3 mM calcium); hCyaAm in 20 mM Hepes, 50 mM NaCl (grey circles ), hCyaAm in 20 mM Hepes, 150 mM NaCl (buffer A, black circles ⦁) and hCyaAm in 20 mM Hepes, 150 mM NaCl, Ficoll 100 g/L (open circles ⚪). Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Error bars: S.D. Three independent preparations of hCyaAm were used for this experiment.
Figure Legend Snippet: Thermal stability of hCyaAm followed by tryptophan fluorescence. Panel (A) Temperature-induced unfolding of hCyaAm at 50 nM followed by intrinsic fluorescence of tryptophan using the ratio of fluorescence emission intensities at 320 nm and 360 nm (rFI 320/360) as described in Materials and Methods. Panel (B) Effect of ionic strength and the presence of the molecular crowding agent Ficoll 100 g/L on the stability of hCyaAm as a function of calcium concentration ( i.e. , 0, 0.2, 0.5, 1, 2 and 3 mM calcium); hCyaAm in 20 mM Hepes, 50 mM NaCl (grey circles ), hCyaAm in 20 mM Hepes, 150 mM NaCl (buffer A, black circles ⦁) and hCyaAm in 20 mM Hepes, 150 mM NaCl, Ficoll 100 g/L (open circles ⚪). Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Error bars: S.D. Three independent preparations of hCyaAm were used for this experiment.

Techniques Used: Fluorescence, Concentration Assay

Hemolytic activity of the different CyaA species. The different CyaA samples, i.e. , CyaA in urea (U-CyaA, green diamonds), holo-CyaA monomers (hCyaAm, red circles), CyaA multimers (M-CyaA, blue squares), were directly diluted into erythrocyte suspensions to reach the indicated final concentrations. Panel (A) Erythrocytes were washed and resuspended in buffer A complemented with 2 mM calcium before the hemolysis assay. CyaA batches were in buffer A complemented with 2 mM calcium. Panel (B) Free calcium was removed from all CyaA samples by buffer exchange on a G25 column equilibrated with buffer A. Cells were also washed and resuspended in buffer A. Panel (C) Erythrocytes were washed in buffer A and resuspended in the presence of 2 mM EDTA. Free calcium was removed from all CyaA samples by buffer exchange on a G25 column equilibrated with buffer A. Panel (D) Excess calcium was removed from a preparation of holo-CyaA monomers by buffer exchange on a G25 column equilibrated with buffer A. The protein (2.6 μM hCyaAm) was then incubated at room temperature with an excess of 4 mM EDTA for different times, 24 hours (filled red circles), 6 hours (filled green circles), 5 hours (filled dark blue circles), 4 hours (filled dark green circles), 3 hours (filled cyan circles), 2 hours (filled violet circles), 1 hour (filled orange circles), 5 min (filled grey circles), or 0 min (plain black circles) and then directly diluted - to the final indicated concentrations - into erythrocytes, washed in Buffer A and resuspended in buffer A +4 mM EDTA. The hemolysis was recorded after an overnight incubation at 37 °C. The insert shows the % of hemolysis (measured at 20 nM final concentration of protein) as a function of time of incubation of hCyaAm with EDTA. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Standard deviation values: ±5%. Two independent preparations of CyaA were used for this experiment.
Figure Legend Snippet: Hemolytic activity of the different CyaA species. The different CyaA samples, i.e. , CyaA in urea (U-CyaA, green diamonds), holo-CyaA monomers (hCyaAm, red circles), CyaA multimers (M-CyaA, blue squares), were directly diluted into erythrocyte suspensions to reach the indicated final concentrations. Panel (A) Erythrocytes were washed and resuspended in buffer A complemented with 2 mM calcium before the hemolysis assay. CyaA batches were in buffer A complemented with 2 mM calcium. Panel (B) Free calcium was removed from all CyaA samples by buffer exchange on a G25 column equilibrated with buffer A. Cells were also washed and resuspended in buffer A. Panel (C) Erythrocytes were washed in buffer A and resuspended in the presence of 2 mM EDTA. Free calcium was removed from all CyaA samples by buffer exchange on a G25 column equilibrated with buffer A. Panel (D) Excess calcium was removed from a preparation of holo-CyaA monomers by buffer exchange on a G25 column equilibrated with buffer A. The protein (2.6 μM hCyaAm) was then incubated at room temperature with an excess of 4 mM EDTA for different times, 24 hours (filled red circles), 6 hours (filled green circles), 5 hours (filled dark blue circles), 4 hours (filled dark green circles), 3 hours (filled cyan circles), 2 hours (filled violet circles), 1 hour (filled orange circles), 5 min (filled grey circles), or 0 min (plain black circles) and then directly diluted - to the final indicated concentrations - into erythrocytes, washed in Buffer A and resuspended in buffer A +4 mM EDTA. The hemolysis was recorded after an overnight incubation at 37 °C. The insert shows the % of hemolysis (measured at 20 nM final concentration of protein) as a function of time of incubation of hCyaAm with EDTA. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Standard deviation values: ±5%. Two independent preparations of CyaA were used for this experiment.

Techniques Used: Activity Assay, Hemolysis Assay, Buffer Exchange, Incubation, Concentration Assay, Standard Deviation

Calcium-dependent stability of hCyaAm over time. Panel (A) (buffer A complemented with 2 mM calcium) and Panel (B) (buffer A complemented with 0.2 mM EDTA): Chromatograms of hCyaAm samples at various time points showing the distribution of monomers and multimers. Samples of hCyaAm at 1 μM were loaded into the injection loop of an Äkta Pure Chromatography System (see Figure S2 ). At various time points, aliquots of CyaA were injected into a Superdex 200 10/300 column equilibrated with the same buffer as the hCyaAm sample loaded into the injection loop. The species of CyaA are defined according to their retention volumes, i.e. , multimers (8–9 mL) and monomers (11–13 mL). Panel (C) fractions of the monomers (filled circles) and multimers (open squares) were calculated by integrating the area under each peak on the chromatograms. SEC experiments were done in buffer A alone (green) or complemented with 2 (red), 0.5 (blue), 0.2 (black) mM calcium. Panel (D) hCyaAm samples were buffer exchanged on G25 column against buffer A complemented with 0.2 mM EDTA and the Superdex 200 10/300 column was equilibrated with the same buffer. Fractions of CyaA monomers (red circles) and CyaA multimers (blue squares) are shown. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Standard deviation values: ±5%. Three independent preparations of hCyaAm were used for this experiment.
Figure Legend Snippet: Calcium-dependent stability of hCyaAm over time. Panel (A) (buffer A complemented with 2 mM calcium) and Panel (B) (buffer A complemented with 0.2 mM EDTA): Chromatograms of hCyaAm samples at various time points showing the distribution of monomers and multimers. Samples of hCyaAm at 1 μM were loaded into the injection loop of an Äkta Pure Chromatography System (see Figure S2 ). At various time points, aliquots of CyaA were injected into a Superdex 200 10/300 column equilibrated with the same buffer as the hCyaAm sample loaded into the injection loop. The species of CyaA are defined according to their retention volumes, i.e. , multimers (8–9 mL) and monomers (11–13 mL). Panel (C) fractions of the monomers (filled circles) and multimers (open squares) were calculated by integrating the area under each peak on the chromatograms. SEC experiments were done in buffer A alone (green) or complemented with 2 (red), 0.5 (blue), 0.2 (black) mM calcium. Panel (D) hCyaAm samples were buffer exchanged on G25 column against buffer A complemented with 0.2 mM EDTA and the Superdex 200 10/300 column was equilibrated with the same buffer. Fractions of CyaA monomers (red circles) and CyaA multimers (blue squares) are shown. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Standard deviation values: ±5%. Three independent preparations of hCyaAm were used for this experiment.

Techniques Used: Injection, Chromatography, Size-exclusion Chromatography, Standard Deviation

Intoxication activity of the different CyaA species. The protein samples, i.e. , hCyaAm (red circles), U-CyaA, (green diamonds), M-CyaA, (blue squares), AC364 (black circles) were directly diluted into erythrocyte suspensions to reach the final concentrations. CyaA in 6 M urea was buffer exchanged on a G25 equilibrated with buffer A +2 mM CaCl 2 , providing the U-CyaA sample. Panel (A) Erythrocytes were washed and resuspended in buffer A complemented with 2 mM calcium. Panel (B) All protein samples were buffer exchanged on a G25 equilibrated with buffer A to remove calcium. Erythrocytes where washed and resuspended in buffer A. Panel (C) cAMP accumulation in erythrocytes as a function of calcium concentration. Erythrocytes were extensively washed in buffer A and then supplemented with the indicated concentrations of calcium ( i.e. , 0, 0.2, 0.5, 0,8, 1.5 and 2 mM CaCl 2 ). Monomeric hCyaAm was desalted on G25 equilibrated in buffer A and diluted into the erythrocyte suspensions at a protein concentration of 2.8 nM, i.e. , 500 ng/mL. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Standard deviation values: ±12 nM cAMP. Three independent preparations of CyaA were used for this experiment.
Figure Legend Snippet: Intoxication activity of the different CyaA species. The protein samples, i.e. , hCyaAm (red circles), U-CyaA, (green diamonds), M-CyaA, (blue squares), AC364 (black circles) were directly diluted into erythrocyte suspensions to reach the final concentrations. CyaA in 6 M urea was buffer exchanged on a G25 equilibrated with buffer A +2 mM CaCl 2 , providing the U-CyaA sample. Panel (A) Erythrocytes were washed and resuspended in buffer A complemented with 2 mM calcium. Panel (B) All protein samples were buffer exchanged on a G25 equilibrated with buffer A to remove calcium. Erythrocytes where washed and resuspended in buffer A. Panel (C) cAMP accumulation in erythrocytes as a function of calcium concentration. Erythrocytes were extensively washed in buffer A and then supplemented with the indicated concentrations of calcium ( i.e. , 0, 0.2, 0.5, 0,8, 1.5 and 2 mM CaCl 2 ). Monomeric hCyaAm was desalted on G25 equilibrated in buffer A and diluted into the erythrocyte suspensions at a protein concentration of 2.8 nM, i.e. , 500 ng/mL. Buffer A contains 20 mM Hepes, 150 mM NaCl, pH 7.4. Standard deviation values: ±12 nM cAMP. Three independent preparations of CyaA were used for this experiment.

Techniques Used: Activity Assay, Concentration Assay, Protein Concentration, Standard Deviation

54) Product Images from "The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats"

Article Title: The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1372

Mutations within the TPR domain of sNASP abolish H3 peptide binding. ( A ) Alignment of the TPR motifs from NASP homologues that retained the H3 116–135 interaction, displayed alongside secondary structural elements and the TPR repeat motif consensus. The positions predicted to face the concave binding channel of the TPR domain fold are indicated by arrows. Asterisks indicate two conserved clusters of residues that are outside of the TPR consensus. Residue numbers for the human homologue are given. The junction of the reconstituted TPR2 is shown as a vertical black line. ( B ) Pulldown experiments of two triple mutants corresponding to the conserved residues identified in (A) under conditions of low stringency (in 20 mM Hepes-KOH pH 7.5, 200 mM sodium chloride) and high stringency (50 mM potassium hydrogen phosphate pH 7.5, 300 mM sodium chloride, 0.1% NP40 and 20% imidazole). ( C ) F2H analysis of GFP-sNASP mutants E211A, E215A, E217A (EEE > AAA) and E246A, Y249S, L253S (EYL > ASS) with mCherry-LacI-H3 or H3 116–135 as indicated. ( D ) Quantification of the enrichment ratio for F2H pairs shown in (C). As comparisons, values for GFP-sNASP recruitment to mCherry-LacI, mCherry-LacI-H3 and mCherry-LacI-H3 116–135 from Figure 1C are shown alongside. Boxes represent the lower quartile, median and upper quartile. Whiskers represent 1.5× the interquartile range. Outliers are indicated by circles. Asterisks indicate a significant difference in enrichment compared to the empty vector control ( P -value
Figure Legend Snippet: Mutations within the TPR domain of sNASP abolish H3 peptide binding. ( A ) Alignment of the TPR motifs from NASP homologues that retained the H3 116–135 interaction, displayed alongside secondary structural elements and the TPR repeat motif consensus. The positions predicted to face the concave binding channel of the TPR domain fold are indicated by arrows. Asterisks indicate two conserved clusters of residues that are outside of the TPR consensus. Residue numbers for the human homologue are given. The junction of the reconstituted TPR2 is shown as a vertical black line. ( B ) Pulldown experiments of two triple mutants corresponding to the conserved residues identified in (A) under conditions of low stringency (in 20 mM Hepes-KOH pH 7.5, 200 mM sodium chloride) and high stringency (50 mM potassium hydrogen phosphate pH 7.5, 300 mM sodium chloride, 0.1% NP40 and 20% imidazole). ( C ) F2H analysis of GFP-sNASP mutants E211A, E215A, E217A (EEE > AAA) and E246A, Y249S, L253S (EYL > ASS) with mCherry-LacI-H3 or H3 116–135 as indicated. ( D ) Quantification of the enrichment ratio for F2H pairs shown in (C). As comparisons, values for GFP-sNASP recruitment to mCherry-LacI, mCherry-LacI-H3 and mCherry-LacI-H3 116–135 from Figure 1C are shown alongside. Boxes represent the lower quartile, median and upper quartile. Whiskers represent 1.5× the interquartile range. Outliers are indicated by circles. Asterisks indicate a significant difference in enrichment compared to the empty vector control ( P -value

Techniques Used: Binding Assay, Plasmid Preparation

55) Product Images from "Structural basis of tubulin recruitment and assembly by microtubule polymerases with tumor overexpressed gene (TOG) domain arrays"

Article Title: Structural basis of tubulin recruitment and assembly by microtubule polymerases with tumor overexpressed gene (TOG) domain arrays

Journal: eLife

doi: 10.7554/eLife.38922

Inactivating interfaces 1 and 2 destabilizes the TOG square organization but does not influence αβ-tubulin-binding activity. ( A ) Purification of INT1 mutant. Top, SEC-elution profile and trace for INT1 showing that it behaves homogenously similarly to wt-Alp14-dimer. Bottom panel show SDS-PAGE for SEC purified fractions. ( B ) Purification of INT2 mutant. Top, SEC-elution profile and trace for INT2 showing that it behaves homogenously similarly to wt-Alp14-dimer. Bottom panel shows SDS-PAGE of SEC purified fractions. ( C ) Purification of INT1 +2 mutant. Top, SEC-elution profile and trace for INT1 +2 showing that it behaves homogenously similarly to wt-Alp14-dimer. Bottom panel shows SDS-PAGE of SEC purified fractions. ( D ) SEC traces for INT1:αβ-tubulin complexes at 100 (light pink) and 200 mM KCl (dark pink) at 2:4 molar ratio showing that inactivating the TOG square assembly has little effect on αβ-tubulin binding. Note that roughly half the αβ-tubulin dissociates at 200 mM KCl due to rapid exchange by TOG2. SDS-PAGE for fractions are shown below. ( E ) SEC traces for INT2:αβ-tubulin complexes at 100 (light green) and 200 mM KCl (dark green) at 2:4 molar ratio showing that inactivating the TOG square assembly has little effect on αβ-tubulin binding. Note that roughly half the αβ-tubulin dissociates at 200 mM KCl due to rapid exchange by TOG2. SDS-PAGE for fractions are shown below. ( F ) SEC traces for INT1 +2:αβ-tubulin complexes at 100 (light grey) and 200 mM KCl (dark grey) at 2:4 molar ratio showing that inactivating the TOG square assembly has little effect on αβ-tubulin binding. Note that roughly half the αβ-tubulin dissociates at 200 mM KCl due to rapid exchange by TOG2. SDS-PAGE for fractions are shown below.
Figure Legend Snippet: Inactivating interfaces 1 and 2 destabilizes the TOG square organization but does not influence αβ-tubulin-binding activity. ( A ) Purification of INT1 mutant. Top, SEC-elution profile and trace for INT1 showing that it behaves homogenously similarly to wt-Alp14-dimer. Bottom panel show SDS-PAGE for SEC purified fractions. ( B ) Purification of INT2 mutant. Top, SEC-elution profile and trace for INT2 showing that it behaves homogenously similarly to wt-Alp14-dimer. Bottom panel shows SDS-PAGE of SEC purified fractions. ( C ) Purification of INT1 +2 mutant. Top, SEC-elution profile and trace for INT1 +2 showing that it behaves homogenously similarly to wt-Alp14-dimer. Bottom panel shows SDS-PAGE of SEC purified fractions. ( D ) SEC traces for INT1:αβ-tubulin complexes at 100 (light pink) and 200 mM KCl (dark pink) at 2:4 molar ratio showing that inactivating the TOG square assembly has little effect on αβ-tubulin binding. Note that roughly half the αβ-tubulin dissociates at 200 mM KCl due to rapid exchange by TOG2. SDS-PAGE for fractions are shown below. ( E ) SEC traces for INT2:αβ-tubulin complexes at 100 (light green) and 200 mM KCl (dark green) at 2:4 molar ratio showing that inactivating the TOG square assembly has little effect on αβ-tubulin binding. Note that roughly half the αβ-tubulin dissociates at 200 mM KCl due to rapid exchange by TOG2. SDS-PAGE for fractions are shown below. ( F ) SEC traces for INT1 +2:αβ-tubulin complexes at 100 (light grey) and 200 mM KCl (dark grey) at 2:4 molar ratio showing that inactivating the TOG square assembly has little effect on αβ-tubulin binding. Note that roughly half the αβ-tubulin dissociates at 200 mM KCl due to rapid exchange by TOG2. SDS-PAGE for fractions are shown below.

Techniques Used: Binding Assay, Activity Assay, Purification, Mutagenesis, Size-exclusion Chromatography, SDS Page

The Alp14 TOG array:αβ-tubulin binding capacities reveal non-equivalent behavior of TOG1 and TOG2 in different conditions. We determined the molar ratio for four Alp14 constructs (described in Figure 1 ) in binding to soluble αβ-tubulins at 100 and 200 mM KCl conditions using quantitative-size exclusion chromatography (SEC). Values for molar ratios are reported in Figure 1D and Table 1 revealing the molar ratios of αβ-tubulin bound to these Alp14 constructs. In each condition, 1 μmol of each Alp14 construct was mixed with a defined αβ-tubulin amount (1 μmol per Alp14 subunit) in each condition, and then analyzed by SEC as shown in A, D, G, and J. The SDS-PAGE results for fractions are shown in B, E, H, and K. ( A, D, G, J ) Top, models for Alp14 constructs studied for tubulin binding using SEC: ( A ) Alp4-monomer (residues 1–510), ( B ) wt-Alp14-dimer (residues 1–690), ( C ) TOG1M (TOG1-inactivated Alp14-dimer mutant), and ( D ) TOG2M (TOG2-inactivated Alp14-dimer mutant). Bottom, SEC-elution profiles for these constructs in complex with αβ-tubulin at 80–100 and 200 mM KCl at the ratios of TOG array subunit:αβ-tubulin described for each condition. Note that the largest Alp14:αβ-tubulin complexes are observed at 100 mM KCl in 1:2 molar ratio for Alp14-monomer, and 2:4 molar ratio for wt-Alp14-dimer constructs. Molar ratios are shown and colored according to each SEC trace. ( B, E, H, K ) Compositions of SEC fractions shown in each panel above ( A, D, G, and J ) using SDS-PAGE. Molar ratios for Alp14:αβ-tubulin are shown in the same colors as the traces above. The SDS-PAGE analyses reveal that αβ-tubulin dissociates at 200 mM KCl, forming a second peak due to dissociation at 200 mM KCl. Note that TOG1M fully dissociates from αβ-tubulin at 200 mM, while TOG2M remains mostly bound to αβ-tubulin at 200 mM KCl. Molar ratios of Alp14 to αβ-tubulin were determined using quantitative densitometry at those conditions and reported in Table 1 and Figure 1D . ( C, F, I, L ) Schematic models, similar to Figure 1F , for Alp14-monomer, wt-Alp14-dimer, TOG1M, and TOG2M in binding αβ-tubulins at 100 mM and 200 mM KCl, revealing the non-equivalent behavior of TOG1 and TOG2 domains in binding and releasing αβ-tubulins in response to different ionic strengths.
Figure Legend Snippet: The Alp14 TOG array:αβ-tubulin binding capacities reveal non-equivalent behavior of TOG1 and TOG2 in different conditions. We determined the molar ratio for four Alp14 constructs (described in Figure 1 ) in binding to soluble αβ-tubulins at 100 and 200 mM KCl conditions using quantitative-size exclusion chromatography (SEC). Values for molar ratios are reported in Figure 1D and Table 1 revealing the molar ratios of αβ-tubulin bound to these Alp14 constructs. In each condition, 1 μmol of each Alp14 construct was mixed with a defined αβ-tubulin amount (1 μmol per Alp14 subunit) in each condition, and then analyzed by SEC as shown in A, D, G, and J. The SDS-PAGE results for fractions are shown in B, E, H, and K. ( A, D, G, J ) Top, models for Alp14 constructs studied for tubulin binding using SEC: ( A ) Alp4-monomer (residues 1–510), ( B ) wt-Alp14-dimer (residues 1–690), ( C ) TOG1M (TOG1-inactivated Alp14-dimer mutant), and ( D ) TOG2M (TOG2-inactivated Alp14-dimer mutant). Bottom, SEC-elution profiles for these constructs in complex with αβ-tubulin at 80–100 and 200 mM KCl at the ratios of TOG array subunit:αβ-tubulin described for each condition. Note that the largest Alp14:αβ-tubulin complexes are observed at 100 mM KCl in 1:2 molar ratio for Alp14-monomer, and 2:4 molar ratio for wt-Alp14-dimer constructs. Molar ratios are shown and colored according to each SEC trace. ( B, E, H, K ) Compositions of SEC fractions shown in each panel above ( A, D, G, and J ) using SDS-PAGE. Molar ratios for Alp14:αβ-tubulin are shown in the same colors as the traces above. The SDS-PAGE analyses reveal that αβ-tubulin dissociates at 200 mM KCl, forming a second peak due to dissociation at 200 mM KCl. Note that TOG1M fully dissociates from αβ-tubulin at 200 mM, while TOG2M remains mostly bound to αβ-tubulin at 200 mM KCl. Molar ratios of Alp14 to αβ-tubulin were determined using quantitative densitometry at those conditions and reported in Table 1 and Figure 1D . ( C, F, I, L ) Schematic models, similar to Figure 1F , for Alp14-monomer, wt-Alp14-dimer, TOG1M, and TOG2M in binding αβ-tubulins at 100 mM and 200 mM KCl, revealing the non-equivalent behavior of TOG1 and TOG2 domains in binding and releasing αβ-tubulins in response to different ionic strengths.

Techniques Used: Binding Assay, Construct, Size-exclusion Chromatography, SDS Page, Mutagenesis

Molar ratios of Alp14 constructs binding to αβ-tubulin binding in the presence of Darpin-D1 (DRP) show no change in stoichiometry, and control SEC-MALS traces. For each condition, 1 μmol of each Alp14 construct (described in Figure 1 ) was mixed with defined molar amount of soluble αβ-tubulin (1–2 μmol moles per Alp14 subunit) and Darpin-D1 (DRP). ( A, D ) Top shows models for DRP (yellow) binding to Alp14 constructs: ( A ) Alp14-monomer (1-510) and (D) wt-Alp14-dimer (residues 1–690). Bottom, SEC elution profiles for Alp14 constructs in complex with αβ-tubulin at 80–100 and 200 mM KCl at two molar ratios described for each condition. Molar ratios are shown and colored according to each SEC trace. Colors for each of the traces match the ratios reported at the top left of each SDS-PAGE panel described below. Note that αβ-tubulin dissociates into a second peak in the 200 mM KCl condition, similar to conditions without DRP described in Figure 1—figure supplement 1 ( Figure 1—figure supplement 1 ). ( B, E ) SDS-PAGE of the four SEC traces shown in A and D, respectively: Alp14 monomer:αβ-tubulin ( B ) and Alp14-dimer:αβ-tubulin complexes ( E ). Top left corner shows the molar ratio in the same color as the trace above. Note that DRP binds αβ-tubulins in each Alp14:αβ-tubulin complex, and that its stoichiometry matches αβ-tubulin. Note the dissociation of αβ-tubulin-DRP from Alp14 constructs, which forms a second peak at 200 mM KCl. ( C, F ) Schematic models for Alp14-monomer and Alp14-dimer in binding αβ-tubulins at 80–100 mM and 200 mM KCl, revealing the non-equivalent behavior of TOG1 and TOG2 domains in releasing αβ-tubulins. DRP can access each β-tubulin interface, suggesting that TOG arrays maintain αβ-tubulins in a non-polymerized state upon binding. To find the effect of DRP binding to Alp14: β-tubulin complex, we determined the molar ratios for two Alp14 constructs binding to αβ-tubulins at 100 and 200 mM KCl conditions in the presence of DRP by quantitative-SEC using panels B and F. The values for molar ratios are reported in Figure 1D and Table 1 , revealing molar ratios of αβ-tubulin bound to two Alp14 constructs were not influenced by DRP binding to αβ-tubulin. This finding suggests that αβ-tubulin binds to TOG arrays in a dimeric and non-polymerized state initially. ( G ) SEC-MALS traces for isolated Alp14-monomer, αβ-tubulin, and wt-Alp14-dimer revealing their masses at 200 mM KCl. These data are summarized in Table 2 . Alp14-monomer is indeed monomeric, while wt-Alp14-dimer is homodimeric and αβ-tubulin is a heterodimer. SEC-MALS measured masses are reported in Table 2 . ( H ) SEC-MALS traces for Alp14-dimer binding to αβ-tubulin at a 2:2 molar ratio with 100 mM KCl or 200 mM KCl, respectively. These data are summarized in Table 2 .
Figure Legend Snippet: Molar ratios of Alp14 constructs binding to αβ-tubulin binding in the presence of Darpin-D1 (DRP) show no change in stoichiometry, and control SEC-MALS traces. For each condition, 1 μmol of each Alp14 construct (described in Figure 1 ) was mixed with defined molar amount of soluble αβ-tubulin (1–2 μmol moles per Alp14 subunit) and Darpin-D1 (DRP). ( A, D ) Top shows models for DRP (yellow) binding to Alp14 constructs: ( A ) Alp14-monomer (1-510) and (D) wt-Alp14-dimer (residues 1–690). Bottom, SEC elution profiles for Alp14 constructs in complex with αβ-tubulin at 80–100 and 200 mM KCl at two molar ratios described for each condition. Molar ratios are shown and colored according to each SEC trace. Colors for each of the traces match the ratios reported at the top left of each SDS-PAGE panel described below. Note that αβ-tubulin dissociates into a second peak in the 200 mM KCl condition, similar to conditions without DRP described in Figure 1—figure supplement 1 ( Figure 1—figure supplement 1 ). ( B, E ) SDS-PAGE of the four SEC traces shown in A and D, respectively: Alp14 monomer:αβ-tubulin ( B ) and Alp14-dimer:αβ-tubulin complexes ( E ). Top left corner shows the molar ratio in the same color as the trace above. Note that DRP binds αβ-tubulins in each Alp14:αβ-tubulin complex, and that its stoichiometry matches αβ-tubulin. Note the dissociation of αβ-tubulin-DRP from Alp14 constructs, which forms a second peak at 200 mM KCl. ( C, F ) Schematic models for Alp14-monomer and Alp14-dimer in binding αβ-tubulins at 80–100 mM and 200 mM KCl, revealing the non-equivalent behavior of TOG1 and TOG2 domains in releasing αβ-tubulins. DRP can access each β-tubulin interface, suggesting that TOG arrays maintain αβ-tubulins in a non-polymerized state upon binding. To find the effect of DRP binding to Alp14: β-tubulin complex, we determined the molar ratios for two Alp14 constructs binding to αβ-tubulins at 100 and 200 mM KCl conditions in the presence of DRP by quantitative-SEC using panels B and F. The values for molar ratios are reported in Figure 1D and Table 1 , revealing molar ratios of αβ-tubulin bound to two Alp14 constructs were not influenced by DRP binding to αβ-tubulin. This finding suggests that αβ-tubulin binds to TOG arrays in a dimeric and non-polymerized state initially. ( G ) SEC-MALS traces for isolated Alp14-monomer, αβ-tubulin, and wt-Alp14-dimer revealing their masses at 200 mM KCl. These data are summarized in Table 2 . Alp14-monomer is indeed monomeric, while wt-Alp14-dimer is homodimeric and αβ-tubulin is a heterodimer. SEC-MALS measured masses are reported in Table 2 . ( H ) SEC-MALS traces for Alp14-dimer binding to αβ-tubulin at a 2:2 molar ratio with 100 mM KCl or 200 mM KCl, respectively. These data are summarized in Table 2 .

Techniques Used: Construct, Binding Assay, Size-exclusion Chromatography, SDS Page, Isolation

56) Product Images from "Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature"

Article Title: Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201704061

PI 3-kinase inhibitors decrease the number of actin comet tails in OCRL-deficient cells and overall model. (A) Purified mCherry-2×FYVE domain used to stain for PI(3)P in cells fixed after treatment with DMSO control, 2 µM wortmannin, or 10 µM Vps34-IN1 in serum-free media for 1 h before imaging. (B) Representative images from time-lapse videos of OCRL-deficient cells expressing F-tractin and treatment with DMSO or the inhibitors, as shown in Video 6. Actin comets were reduced on PI 3-kinase inhibitor treatment. Bars, 10 µm. (C) Quantification of actin comets from n = 30 cell regions for each treatment. Bars indicate the mean ± SEM for each condition. Difference assessed by ordinary one-way ANOVA with Dunnett’s multiple comparisons test, overall ANOVA, and comparing each inhibitor treatment to DMSO control cells. ***, P ≤ 0.0001. (D) Pathway of curvature signaling to actin polymerization via PI(4,5)P 2 /PI(3)P/SNX9 during CME. High curvature activates a cascade of phosphoinositol metabolism to change membrane identity and trigger actin polymerization at PI(4,5)P 2 /PI(3)P-enriched and highly curved sites via the oligo merization of SNX9 during endocytosis. In Lowe syndrome, OCRL deficiency increases PI(4,5)P 2 levels on PI(3)P intermediates, triggering SNX9 assembly. PI(4,5)P 2 also activates Cdc42 (independently of the curvature) for the relief of N-WASP inhibition. N-WASP oligomerization (via SNX9 binding) finally drives superactivation of the Arp2/3 complex and, thus, actin polymerization.
Figure Legend Snippet: PI 3-kinase inhibitors decrease the number of actin comet tails in OCRL-deficient cells and overall model. (A) Purified mCherry-2×FYVE domain used to stain for PI(3)P in cells fixed after treatment with DMSO control, 2 µM wortmannin, or 10 µM Vps34-IN1 in serum-free media for 1 h before imaging. (B) Representative images from time-lapse videos of OCRL-deficient cells expressing F-tractin and treatment with DMSO or the inhibitors, as shown in Video 6. Actin comets were reduced on PI 3-kinase inhibitor treatment. Bars, 10 µm. (C) Quantification of actin comets from n = 30 cell regions for each treatment. Bars indicate the mean ± SEM for each condition. Difference assessed by ordinary one-way ANOVA with Dunnett’s multiple comparisons test, overall ANOVA, and comparing each inhibitor treatment to DMSO control cells. ***, P ≤ 0.0001. (D) Pathway of curvature signaling to actin polymerization via PI(4,5)P 2 /PI(3)P/SNX9 during CME. High curvature activates a cascade of phosphoinositol metabolism to change membrane identity and trigger actin polymerization at PI(4,5)P 2 /PI(3)P-enriched and highly curved sites via the oligo merization of SNX9 during endocytosis. In Lowe syndrome, OCRL deficiency increases PI(4,5)P 2 levels on PI(3)P intermediates, triggering SNX9 assembly. PI(4,5)P 2 also activates Cdc42 (independently of the curvature) for the relief of N-WASP inhibition. N-WASP oligomerization (via SNX9 binding) finally drives superactivation of the Arp2/3 complex and, thus, actin polymerization.

Techniques Used: Purification, Staining, Imaging, Expressing, Inhibition, Binding Assay

Identifying the minimal machinery required for SNX9-regulated actin assembly. (A) Maximal activation of actin polymerization by liposomes containing 4% PI(4,5)P 2 + 1% PI(3)P (with 48% PC and 47% PS) assayed by pyrene actin assay in the minimal reconstituted system (20 nM Arp2/3 complex, 500 nM Cdc42⋅GTP-γS, 100 nM N-WASP/WIP complex, 100 nM SNX9, and 1 µM actin, 65:35 pyrene actin) compared with 4% PI(4,5)P 2 alone, actin alone, and activation by GST-VCA fragment from N-WASP as a positive control. Data show the mean of eight traces. AU, arbitrary units. (B) Maximal rates from two technical repeats each of four independent experiments showing the mean and SEM. The maximal rates were normalized against 100% activation by GST-VCA. Significance was tested using an ANOVA test with a Tukey's multiple comparison post-hoc test; actin versus PI(4,5)P 2 : P = 0.900; actin versus PI(4,5)P 2 /PI(3)P: ***, P = 0.001; actin versus −Cdc42: P = 0.9000. ns, not significant. (C) Direct observation of liposomes (4% PI(4,5)P 2 , 1% PI(3)P, 65% PC, 30% PS; purple) in the presence of the minimal purified system containing 50 nM Arp2/3 complex, 50 nM Cdc42⋅GTP-γS, 100 nM N-WASP–WIP complex, 100 nM SNX9, 8 µM unlabeled actin, and 0.3 µM Alexa Fluor 647–labeled actin. Actin asters form at the surface of highly curved liposomes only when all components are present. (D) Activation of Cdc42 is needed. Direct observation of liposome samples with a continuous size distribution from 50 nm to 5 µm (4% PI(4,5)P 2 , 1% PI(3)P, 65% PC, 30% PS; purple) in the presence of the minimal purified system containing Cdc42⋅GDP. (C and D) Bars, 3 µm. (E) Electron micrograph of actin asters after incubation of PI(4,5)P 2 /PI(3)P liposomes with the minimal purified system shows disordered and branched actin filaments. Bar, 100 nm. (F–I) All components of the purified system are required for efficient actin polymerization. No actin polymerization is seen with the minimal purified system minus each individual component: Cdc42⋅GTP-γS (F), SNX9 (G), N-WASP–WIP (H), or Arp2/3 complex (I). Bars, 6 µm.
Figure Legend Snippet: Identifying the minimal machinery required for SNX9-regulated actin assembly. (A) Maximal activation of actin polymerization by liposomes containing 4% PI(4,5)P 2 + 1% PI(3)P (with 48% PC and 47% PS) assayed by pyrene actin assay in the minimal reconstituted system (20 nM Arp2/3 complex, 500 nM Cdc42⋅GTP-γS, 100 nM N-WASP/WIP complex, 100 nM SNX9, and 1 µM actin, 65:35 pyrene actin) compared with 4% PI(4,5)P 2 alone, actin alone, and activation by GST-VCA fragment from N-WASP as a positive control. Data show the mean of eight traces. AU, arbitrary units. (B) Maximal rates from two technical repeats each of four independent experiments showing the mean and SEM. The maximal rates were normalized against 100% activation by GST-VCA. Significance was tested using an ANOVA test with a Tukey's multiple comparison post-hoc test; actin versus PI(4,5)P 2 : P = 0.900; actin versus PI(4,5)P 2 /PI(3)P: ***, P = 0.001; actin versus −Cdc42: P = 0.9000. ns, not significant. (C) Direct observation of liposomes (4% PI(4,5)P 2 , 1% PI(3)P, 65% PC, 30% PS; purple) in the presence of the minimal purified system containing 50 nM Arp2/3 complex, 50 nM Cdc42⋅GTP-γS, 100 nM N-WASP–WIP complex, 100 nM SNX9, 8 µM unlabeled actin, and 0.3 µM Alexa Fluor 647–labeled actin. Actin asters form at the surface of highly curved liposomes only when all components are present. (D) Activation of Cdc42 is needed. Direct observation of liposome samples with a continuous size distribution from 50 nm to 5 µm (4% PI(4,5)P 2 , 1% PI(3)P, 65% PC, 30% PS; purple) in the presence of the minimal purified system containing Cdc42⋅GDP. (C and D) Bars, 3 µm. (E) Electron micrograph of actin asters after incubation of PI(4,5)P 2 /PI(3)P liposomes with the minimal purified system shows disordered and branched actin filaments. Bar, 100 nm. (F–I) All components of the purified system are required for efficient actin polymerization. No actin polymerization is seen with the minimal purified system minus each individual component: Cdc42⋅GTP-γS (F), SNX9 (G), N-WASP–WIP (H), or Arp2/3 complex (I). Bars, 6 µm.

Techniques Used: Activation Assay, Pyrene Actin Assay, Positive Control, Purification, Labeling, Incubation

57) Product Images from "Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication"

Article Title: Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky460

Cleavage of mismatch-containing DNA by Cgl EndoMS-WT and Cgl EndoMS-ΔCter with Cgl β-clamp. ( A ) The 5′-Cy5-labeled DNA substrates (5 nM) containing the GT base pair were incubated with the proteins. The products were analyzed by native 10% PAGE followed by laser scanning (cropped gel image). Lanes 1, positive control (20 nM TkoEndoMS, as a monomer); 2, negative control (no proteins); 3–7, 50 nM Cgl EndoMS-WT (as a monomer); 8–12, 50 nM Cgl EndoMS-ΔCter (as a monomer); 13, no EndoMS. The indicated concentrations (as a monomer) of Cgl β-clamp were added to the reactions. Representative results are shown. The band assignments are indicated on the side of the panels: s, substrates: p, cleaved products. ( B ) Quantification of the cleaved products. Independent data points from three measurements are plotted. ( C ) The physical interactions of Cgl EndoMS with Cgl β-clamp were characterized by SPR using a BIACORE J system (Biacore Inc.). Purified Cgl β-Clamp was immobilized on the sensor Chip CM5, and various concentrations (indicated on the right sides of the sensorgrams) of Cgl EndoMS-WT and Cgl EndoMS-ΔCter were loaded onto the chip for 2 min at a flow rate of 30 μl/min in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Regeneration was achieved using a 1-min injection of 0.025% SDS. Each background response was subtracted. Data were analyzed using BIAevaluation v.3 software (Biacore Inc.)
Figure Legend Snippet: Cleavage of mismatch-containing DNA by Cgl EndoMS-WT and Cgl EndoMS-ΔCter with Cgl β-clamp. ( A ) The 5′-Cy5-labeled DNA substrates (5 nM) containing the GT base pair were incubated with the proteins. The products were analyzed by native 10% PAGE followed by laser scanning (cropped gel image). Lanes 1, positive control (20 nM TkoEndoMS, as a monomer); 2, negative control (no proteins); 3–7, 50 nM Cgl EndoMS-WT (as a monomer); 8–12, 50 nM Cgl EndoMS-ΔCter (as a monomer); 13, no EndoMS. The indicated concentrations (as a monomer) of Cgl β-clamp were added to the reactions. Representative results are shown. The band assignments are indicated on the side of the panels: s, substrates: p, cleaved products. ( B ) Quantification of the cleaved products. Independent data points from three measurements are plotted. ( C ) The physical interactions of Cgl EndoMS with Cgl β-clamp were characterized by SPR using a BIACORE J system (Biacore Inc.). Purified Cgl β-Clamp was immobilized on the sensor Chip CM5, and various concentrations (indicated on the right sides of the sensorgrams) of Cgl EndoMS-WT and Cgl EndoMS-ΔCter were loaded onto the chip for 2 min at a flow rate of 30 μl/min in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Regeneration was achieved using a 1-min injection of 0.025% SDS. Each background response was subtracted. Data were analyzed using BIAevaluation v.3 software (Biacore Inc.)

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Positive Control, Negative Control, SPR Assay, Purification, Chromatin Immunoprecipitation, Flow Cytometry, Injection, Software

58) Product Images from "Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP"

Article Title: Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP

Journal: Acta Crystallographica. Section F, Structural Biology Communications

doi: 10.1107/S2053230X13034705

Melting curve. A temperature melting curve of AtzF was performed in triplicate, using differential scanning fluorimetry with the dye SYPRO Orange. The red curves are the AtzF protein (in triplicate) in the buffer used for crystallization (50 m M HEPES, 100 m M NaCl pH 7.5). The blue curves are a 0.1 mg ml −1 lysozyme control.
Figure Legend Snippet: Melting curve. A temperature melting curve of AtzF was performed in triplicate, using differential scanning fluorimetry with the dye SYPRO Orange. The red curves are the AtzF protein (in triplicate) in the buffer used for crystallization (50 m M HEPES, 100 m M NaCl pH 7.5). The blue curves are a 0.1 mg ml −1 lysozyme control.

Techniques Used: Crystallization Assay

59) Product Images from "Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains"

Article Title: Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001921

NT and CT respond differently to lowered pH. Stability of NT and CT (from A. ventricosus ) in (A) 20 mM HEPES/MES buffer with 154 mM NaCl and (B) the same buffer without NaCl, measured with Trp fluorescence and CD spectroscopy at 222 nm, respectively, presented as urea concentrations for apparent half-denaturation ([den] 50% , see Materials and Methods for details on how [den] 50% was determined) as a function of pH. The pH region in which CA activity is found in major ampullate glands is indicated by a shaded area in (A).
Figure Legend Snippet: NT and CT respond differently to lowered pH. Stability of NT and CT (from A. ventricosus ) in (A) 20 mM HEPES/MES buffer with 154 mM NaCl and (B) the same buffer without NaCl, measured with Trp fluorescence and CD spectroscopy at 222 nm, respectively, presented as urea concentrations for apparent half-denaturation ([den] 50% , see Materials and Methods for details on how [den] 50% was determined) as a function of pH. The pH region in which CA activity is found in major ampullate glands is indicated by a shaded area in (A).

Techniques Used: Fluorescence, Spectroscopy, Activity Assay

60) Product Images from "Functional insight into the role of Orc6 in septin complex filament formation in Drosophila"

Article Title: Functional insight into the role of Orc6 in septin complex filament formation in Drosophila

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E14-02-0734

Mutations in septin GTPase domain result in defects of septin filament formation. Recombinant wild-type or mutant septin complex His-Pnut-Sep2-Sep1 (10 ng/μl) was incubated with or without Orc6 (15 ng/μl) in the absence or presence of 300 μM GTP as indicated. Wild-type septin complex (A) or complexes containing GTPase mutant Pnut(G4) (B), GTPase mutant Sep1(G4) (C), or triple GTPase mutant Sep2(G1,G3,G4) (D) were used. Samples were incubated for 2 h at 22°C and then visualized by negative-stain transmission EM. Scale bar, 100 nm.
Figure Legend Snippet: Mutations in septin GTPase domain result in defects of septin filament formation. Recombinant wild-type or mutant septin complex His-Pnut-Sep2-Sep1 (10 ng/μl) was incubated with or without Orc6 (15 ng/μl) in the absence or presence of 300 μM GTP as indicated. Wild-type septin complex (A) or complexes containing GTPase mutant Pnut(G4) (B), GTPase mutant Sep1(G4) (C), or triple GTPase mutant Sep2(G1,G3,G4) (D) were used. Samples were incubated for 2 h at 22°C and then visualized by negative-stain transmission EM. Scale bar, 100 nm.

Techniques Used: Recombinant, Mutagenesis, Incubation, Staining, Transmission Assay

61) Product Images from "Neuronal Calcium Sensor-1 (Ncs1p) Is Up-regulated by Calcineurin to Promote Ca2+ Tolerance in Fission Yeast *"

Article Title: Neuronal Calcium Sensor-1 (Ncs1p) Is Up-regulated by Calcineurin to Promote Ca2+ Tolerance in Fission Yeast *

Journal:

doi: 10.1074/jbc.M109.058594

Electrophoretic mobility shift assay of Prz1p binding to ncs1 promoter sequence. 5 n m of Cy5 labeled duplex DNA (see “Experimental Procedures”) in 10 m m HEPES (pH 7.5), 0.1 m NaCl, and 5 m m Mg 2+ was incubated with buffer blank ( lane 1
Figure Legend Snippet: Electrophoretic mobility shift assay of Prz1p binding to ncs1 promoter sequence. 5 n m of Cy5 labeled duplex DNA (see “Experimental Procedures”) in 10 m m HEPES (pH 7.5), 0.1 m NaCl, and 5 m m Mg 2+ was incubated with buffer blank ( lane 1

Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Labeling, Incubation

62) Product Images from "Ternary complex of Kif2A-bound tandem tubulin heterodimers represents a kinesin-13-mediated microtubule depolymerization reaction intermediate"

Article Title: Ternary complex of Kif2A-bound tandem tubulin heterodimers represents a kinesin-13-mediated microtubule depolymerization reaction intermediate

Journal: Nature Communications

doi: 10.1038/s41467-018-05025-7

Functional analysis of Kif2A constructs. a Domain composition of human Kif2A and schematic of the Kif2A constructs used in this study. The length of the bar is proportional to the number of amino acids. b MT depolymerization activity of Kif2A constructs. Taxol-stabilized MTs were incubated with the indicated concentrations of Kif2A-NM and Kif2A-MD, or no kinesin for 10 min in BRB80 buffer. Free tubulin and MT polymers were separated into supernatant (S) and pellet (P) fractions by ultra-centrifugation-based sedimentation assay. Fractions were resuspended and boiled in Laemmli buffer. Equal portions were loaded and analyzed on a Coomassie blue-stained SDS-PAGE gel. c SEC profiles of Kif2A-NM alone (gray dashed line), tubulin–DARPin complex (black dotted line), and Kif2A-NM–tubulin–DARPin complex in 0.8:1:1 molar ratio (black solid line). All samples were supplemented with 0.1 mM AMPPNP and applied to an Superdex 200 10/300 GL column in HEPES buffer. d Twelve percent SDS-PAGE gels of elution fractions from the above experiments. The molecular weight of Kif2A-NM = 48 kDa, tubulin = 50 kDa, and DARPin = 18 kDa. The inset shows a 10% SDS-PAGE gel of fractions containing the Kif2A-NM–tubulin–DARPin complex in order to visualize the Kif2A and tubulin proteins. All uncropped gels are shown in Supplementary Figures 9, 10, 11, 12, 13 and 14
Figure Legend Snippet: Functional analysis of Kif2A constructs. a Domain composition of human Kif2A and schematic of the Kif2A constructs used in this study. The length of the bar is proportional to the number of amino acids. b MT depolymerization activity of Kif2A constructs. Taxol-stabilized MTs were incubated with the indicated concentrations of Kif2A-NM and Kif2A-MD, or no kinesin for 10 min in BRB80 buffer. Free tubulin and MT polymers were separated into supernatant (S) and pellet (P) fractions by ultra-centrifugation-based sedimentation assay. Fractions were resuspended and boiled in Laemmli buffer. Equal portions were loaded and analyzed on a Coomassie blue-stained SDS-PAGE gel. c SEC profiles of Kif2A-NM alone (gray dashed line), tubulin–DARPin complex (black dotted line), and Kif2A-NM–tubulin–DARPin complex in 0.8:1:1 molar ratio (black solid line). All samples were supplemented with 0.1 mM AMPPNP and applied to an Superdex 200 10/300 GL column in HEPES buffer. d Twelve percent SDS-PAGE gels of elution fractions from the above experiments. The molecular weight of Kif2A-NM = 48 kDa, tubulin = 50 kDa, and DARPin = 18 kDa. The inset shows a 10% SDS-PAGE gel of fractions containing the Kif2A-NM–tubulin–DARPin complex in order to visualize the Kif2A and tubulin proteins. All uncropped gels are shown in Supplementary Figures 9, 10, 11, 12, 13 and 14

Techniques Used: Functional Assay, Construct, Activity Assay, Incubation, Centrifugation, Sedimentation, Staining, SDS Page, Size-exclusion Chromatography, Molecular Weight

Structure of Kif2A-NM in complex with tubulin and DARPin. a The X-ray crystal structure of a Kif2A-NM–tubulin–DARPin complex is shown in two views (from the top and side of the tubulin filament). The Kif2A is in black; α-tubulin is in orange; β-tubulin is in green; and DARPin is in light brown. The insert represents the F obs − F calc omit map (contoured at 3.0 σ ) of the neck of Kif2A-NM calculated after deletion of the neck from final model. All figures of structural models were generated with PyMOL 67 . b SAXS envelopes for the Kif2A-NM–tubulin–DARPin complex. The crystal structure of the complex shown in ribbons representation and superimposed onto envelopes (in gray) by Chimera 68 . c SEC profiles of mixtures of Kif2A-NM, tubulin, and DARPin at the indicated molar ratios. All mixtures were supplemented with 0.1 mM AMPPNP and passed through a Superdex 200 10/300 GL column in HEPES buffer
Figure Legend Snippet: Structure of Kif2A-NM in complex with tubulin and DARPin. a The X-ray crystal structure of a Kif2A-NM–tubulin–DARPin complex is shown in two views (from the top and side of the tubulin filament). The Kif2A is in black; α-tubulin is in orange; β-tubulin is in green; and DARPin is in light brown. The insert represents the F obs − F calc omit map (contoured at 3.0 σ ) of the neck of Kif2A-NM calculated after deletion of the neck from final model. All figures of structural models were generated with PyMOL 67 . b SAXS envelopes for the Kif2A-NM–tubulin–DARPin complex. The crystal structure of the complex shown in ribbons representation and superimposed onto envelopes (in gray) by Chimera 68 . c SEC profiles of mixtures of Kif2A-NM, tubulin, and DARPin at the indicated molar ratios. All mixtures were supplemented with 0.1 mM AMPPNP and passed through a Superdex 200 10/300 GL column in HEPES buffer

Techniques Used: Generated, Size-exclusion Chromatography

63) Product Images from "Ternary complex of Kif2A-bound tandem tubulin heterodimers represents a kinesin-13-mediated microtubule depolymerization reaction intermediate"

Article Title: Ternary complex of Kif2A-bound tandem tubulin heterodimers represents a kinesin-13-mediated microtubule depolymerization reaction intermediate

Journal: Nature Communications

doi: 10.1038/s41467-018-05025-7

Functional analysis of Kif2A constructs. a Domain composition of human Kif2A and schematic of the Kif2A constructs used in this study. The length of the bar is proportional to the number of amino acids. b MT depolymerization activity of Kif2A constructs. Taxol-stabilized MTs were incubated with the indicated concentrations of Kif2A-NM and Kif2A-MD, or no kinesin for 10 min in BRB80 buffer. Free tubulin and MT polymers were separated into supernatant (S) and pellet (P) fractions by ultra-centrifugation-based sedimentation assay. Fractions were resuspended and boiled in Laemmli buffer. Equal portions were loaded and analyzed on a Coomassie blue-stained SDS-PAGE gel. c SEC profiles of Kif2A-NM alone (gray dashed line), tubulin–DARPin complex (black dotted line), and Kif2A-NM–tubulin–DARPin complex in 0.8:1:1 molar ratio (black solid line). All samples were supplemented with 0.1 mM AMPPNP and applied to an Superdex 200 10/300 GL column in HEPES buffer. d Twelve percent SDS-PAGE gels of elution fractions from the above experiments. The molecular weight of Kif2A-NM = 48 kDa, tubulin = 50 kDa, and DARPin = 18 kDa. The inset shows a 10% SDS-PAGE gel of fractions containing the Kif2A-NM–tubulin–DARPin complex in order to visualize the Kif2A and tubulin proteins. All uncropped gels are shown in Supplementary Figures 9, 10, 11, 12, 13 and 14
Figure Legend Snippet: Functional analysis of Kif2A constructs. a Domain composition of human Kif2A and schematic of the Kif2A constructs used in this study. The length of the bar is proportional to the number of amino acids. b MT depolymerization activity of Kif2A constructs. Taxol-stabilized MTs were incubated with the indicated concentrations of Kif2A-NM and Kif2A-MD, or no kinesin for 10 min in BRB80 buffer. Free tubulin and MT polymers were separated into supernatant (S) and pellet (P) fractions by ultra-centrifugation-based sedimentation assay. Fractions were resuspended and boiled in Laemmli buffer. Equal portions were loaded and analyzed on a Coomassie blue-stained SDS-PAGE gel. c SEC profiles of Kif2A-NM alone (gray dashed line), tubulin–DARPin complex (black dotted line), and Kif2A-NM–tubulin–DARPin complex in 0.8:1:1 molar ratio (black solid line). All samples were supplemented with 0.1 mM AMPPNP and applied to an Superdex 200 10/300 GL column in HEPES buffer. d Twelve percent SDS-PAGE gels of elution fractions from the above experiments. The molecular weight of Kif2A-NM = 48 kDa, tubulin = 50 kDa, and DARPin = 18 kDa. The inset shows a 10% SDS-PAGE gel of fractions containing the Kif2A-NM–tubulin–DARPin complex in order to visualize the Kif2A and tubulin proteins. All uncropped gels are shown in Supplementary Figures 9, 10, 11, 12, 13 and 14

Techniques Used: Functional Assay, Construct, Activity Assay, Incubation, Centrifugation, Sedimentation, Staining, SDS Page, Size-exclusion Chromatography, Molecular Weight

64) Product Images from "The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation"

Article Title: The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0089264

Biological activity determination of sCD83. sCD83 bound to the putative CD83 receptor on monocytes but not on T cells. (A and B) PBMC were incubated with soluble CD83 or BSA, and then coated with either PE-anti-CD83 (A) or anti-His followed by FITC-Anti-mouse IgG (B). Cells were then coated with anti-CD3 or anti-CD14 to distinguish the monocyte and T cells. CD14 + monocytes and CD3 + T cells were gated and analysis for CD83 receptor expression. (C) sCD83 suppressed ConA-stimulated PBMC proliferation. PBMCs were stained with CFSE, stimulated with ConA, and cultured with or without 20 µg/mL of sCD83 for the indicated time periods. (D) The means ± the standard error of the mean (SEM) of the MFI from (C) are shown as bar graphs. (E) sCD83 activated the NF-κB pathway. Isolated human PBMCs stimulated by sCD83 (10 µg/mL) were cultured in RPMI 1640 medium that contained 10% fetal bovine sera at 37°C for 12 h. The PBMCs were collected and lysed to detect the activation of the NF-κB pathway using Western blotting.
Figure Legend Snippet: Biological activity determination of sCD83. sCD83 bound to the putative CD83 receptor on monocytes but not on T cells. (A and B) PBMC were incubated with soluble CD83 or BSA, and then coated with either PE-anti-CD83 (A) or anti-His followed by FITC-Anti-mouse IgG (B). Cells were then coated with anti-CD3 or anti-CD14 to distinguish the monocyte and T cells. CD14 + monocytes and CD3 + T cells were gated and analysis for CD83 receptor expression. (C) sCD83 suppressed ConA-stimulated PBMC proliferation. PBMCs were stained with CFSE, stimulated with ConA, and cultured with or without 20 µg/mL of sCD83 for the indicated time periods. (D) The means ± the standard error of the mean (SEM) of the MFI from (C) are shown as bar graphs. (E) sCD83 activated the NF-κB pathway. Isolated human PBMCs stimulated by sCD83 (10 µg/mL) were cultured in RPMI 1640 medium that contained 10% fetal bovine sera at 37°C for 12 h. The PBMCs were collected and lysed to detect the activation of the NF-κB pathway using Western blotting.

Techniques Used: Activity Assay, Incubation, Expressing, Staining, Cell Culture, Isolation, Activation Assay, Western Blot

65) Product Images from "Targeting granzyme B to tumor cells using a yoked human chorionic gonadotropin"

Article Title: Targeting granzyme B to tumor cells using a yoked human chorionic gonadotropin

Journal: Cancer Chemotherapy and Pharmacology

doi: 10.1007/s00280-011-1573-4

a Schematic of the structure of hexahistidine-tagged GrB-YCG. b Commassie blue-stained SDS–PAGE gel run under reducing conditions showing steps in GrB-YCG purification. The gel shows analysis of medium from infected Sf9 cells, and the subsequent steps in purification. Lane 1 protein markers; lane 2 medium from infected Sf9 cells; lane 3 ultrafiltrate from 10 kDa cut-off Hydrosart Microfilter Cassette when concentrating the harvested medium; lane 4 concentrated medium; lane 5 6xHis-tagged GrB-YCG after purification by nickel-NTA metal affinity chromatography; lane 6 rEK-cleaved mixture; lane 7 final purified GrB-YCG. c Western blot analysis of the purified GrB-YCG fusion protein before and after rEK cleavage. d Enzymatic activity of GrB-YCG. The GrB-YCG displayed intact serine protease activity at the same level as human recombinant GrB only after the cleavage by rEK
Figure Legend Snippet: a Schematic of the structure of hexahistidine-tagged GrB-YCG. b Commassie blue-stained SDS–PAGE gel run under reducing conditions showing steps in GrB-YCG purification. The gel shows analysis of medium from infected Sf9 cells, and the subsequent steps in purification. Lane 1 protein markers; lane 2 medium from infected Sf9 cells; lane 3 ultrafiltrate from 10 kDa cut-off Hydrosart Microfilter Cassette when concentrating the harvested medium; lane 4 concentrated medium; lane 5 6xHis-tagged GrB-YCG after purification by nickel-NTA metal affinity chromatography; lane 6 rEK-cleaved mixture; lane 7 final purified GrB-YCG. c Western blot analysis of the purified GrB-YCG fusion protein before and after rEK cleavage. d Enzymatic activity of GrB-YCG. The GrB-YCG displayed intact serine protease activity at the same level as human recombinant GrB only after the cleavage by rEK

Techniques Used: Staining, SDS Page, Purification, Infection, Affinity Chromatography, Western Blot, Activity Assay, Recombinant

66) Product Images from "Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation"

Article Title: Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201205021

Drg1 specifically releases Rlp24 from pre-60S particles. (A) Pre-60S particles from the temperature-sensitive drg1-18 mutant incubated at 37°C were purified with Arx1-TAP as bait and immobilized on calmodulin beads. After incubation with Drg1 in the presence (+) or absence (−) of ATP and the Nup116 fragment N172, supernatants (containing released and unbound proteins) were collected and TCA precipitated. pre-60S particles were eluted from the calmodulin beads and concentrated by TCA precipitation. Supernatants (released) and eluates (pre-60S bound) were analyzed by SDS-PAGE and Coomassie staining, as well as Western blotting. (B) Pre-60S particles purified from the Δnup116 as well as from the nup116Δ110-166 mutant show increased amounts of Drg1 and Rlp24. The nup116Δ strain carrying centromeric plasmids with wild-type NUP116 or the nup116 variant lacking codons 110–166 were grown to late log phase and pre-60S particles were isolated via Arx1-TAP. Purified particles were separated by SDS-PAGE and analyzed for the levels of pre-60S maturation factors by Western blotting.
Figure Legend Snippet: Drg1 specifically releases Rlp24 from pre-60S particles. (A) Pre-60S particles from the temperature-sensitive drg1-18 mutant incubated at 37°C were purified with Arx1-TAP as bait and immobilized on calmodulin beads. After incubation with Drg1 in the presence (+) or absence (−) of ATP and the Nup116 fragment N172, supernatants (containing released and unbound proteins) were collected and TCA precipitated. pre-60S particles were eluted from the calmodulin beads and concentrated by TCA precipitation. Supernatants (released) and eluates (pre-60S bound) were analyzed by SDS-PAGE and Coomassie staining, as well as Western blotting. (B) Pre-60S particles purified from the Δnup116 as well as from the nup116Δ110-166 mutant show increased amounts of Drg1 and Rlp24. The nup116Δ strain carrying centromeric plasmids with wild-type NUP116 or the nup116 variant lacking codons 110–166 were grown to late log phase and pre-60S particles were isolated via Arx1-TAP. Purified particles were separated by SDS-PAGE and analyzed for the levels of pre-60S maturation factors by Western blotting.

Techniques Used: Mutagenesis, Incubation, Purification, TCA Precipitation, SDS Page, Staining, Western Blot, Variant Assay, Isolation

67) Product Images from "Identification of a Chemoreceptor Zinc-Binding Domain Common to Cytoplasmic Bacterial Chemoreceptors ▿"

Article Title: Identification of a Chemoreceptor Zinc-Binding Domain Common to Cytoplasmic Bacterial Chemoreceptors ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.05140-11

ICP-MS analysis of the metal content of TlpD after exposure to the chelator TPEN. A total of 15 μM GST-TlpD in HEPES buffer (or buffer alone) was incubated with 75 to 600 μM TPEN for 48 h at 4°C. The protein was filtered from the
Figure Legend Snippet: ICP-MS analysis of the metal content of TlpD after exposure to the chelator TPEN. A total of 15 μM GST-TlpD in HEPES buffer (or buffer alone) was incubated with 75 to 600 μM TPEN for 48 h at 4°C. The protein was filtered from the

Techniques Used: Mass Spectrometry, Incubation

68) Product Images from "Identification of glycosylated marker proteins of epithelial polarity in MDCK cells by homology driven proteomics"

Article Title: Identification of glycosylated marker proteins of epithelial polarity in MDCK cells by homology driven proteomics

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-7-8

A. WGA lectin affinity chromatography of MDCK cell membrane proteins. Bound proteins were eluted with 0.3 M N-acetylglucosamine, and stained by Coomassie blue after gel electrophoresis. L: aliquot of loaded protein preparation. E: eluted protein pattern (bracket indicates 114 kDa region). B. Flow chart for the purification of gp114. MDCK cell membranes were recovered by high-speed centrifugation from a postmitochondrial supernatant and partially solubilized by treatment with the non-ionic detergent Triton X-100 on ice. Soluble proteins were applied to immunoaffinity columns, and the eluted fractions concentrated by methanol-chloroform extraction-precipitation. Gp114 did not accumulate at the
Figure Legend Snippet: A. WGA lectin affinity chromatography of MDCK cell membrane proteins. Bound proteins were eluted with 0.3 M N-acetylglucosamine, and stained by Coomassie blue after gel electrophoresis. L: aliquot of loaded protein preparation. E: eluted protein pattern (bracket indicates 114 kDa region). B. Flow chart for the purification of gp114. MDCK cell membranes were recovered by high-speed centrifugation from a postmitochondrial supernatant and partially solubilized by treatment with the non-ionic detergent Triton X-100 on ice. Soluble proteins were applied to immunoaffinity columns, and the eluted fractions concentrated by methanol-chloroform extraction-precipitation. Gp114 did not accumulate at the "protein" interface between aqueous and lipid phase but stayed in the hydrophilic supernatant. C. Enrichment of gp114. Lane 1 (A) corresponds to the methanolic phase after chloroform-methanol extraction of eluted proteins from the gp114 immunoaffinity column. Gp114 (arrowhead) is only weakly stained by Coomassie solution. Lane 2 (Ip) contains deglycosylated gp114 (arrowhead) after immunoprecipitation which was confirmed by Western blotting (not shown). The heavy chain of gp114 IgG is indicated by a white arrowhead.

Techniques Used: Whole Genome Amplification, Affinity Chromatography, Staining, Nucleic Acid Electrophoresis, Flow Cytometry, Purification, Centrifugation, Immunoprecipitation, Western Blot

69) Product Images from "A homodimer interface without base pairs in an RNA mimic of red fluorescent protein"

Article Title: A homodimer interface without base pairs in an RNA mimic of red fluorescent protein

Journal: Nature chemical biology

doi: 10.1038/nchembio.2475

Biophysical analysis of Corn-DFHO dimer. ( a ) Non-normalized c (s) distributions for Corn RNA at three concentrations and tRNA Lys3 at a single concentration. ( b ) Fluorescence-size exclusion chromatogram (F-SEC) for the Corn-DFHO complex. Absorbance was monitored at 260 nm and fluorescence at the emission maximum (543 nm). Arrow denotes void volume (8 mL) as determined by an independent run with blue dextran (MW ~ 6 MDa) under identical conditions. Elution volume of the 76 nt tRNA Lys3 (10.9 mL) in an independent experiment under identical conditions is also indicated. ( c ) Job plot 23 for DFHO binding to Corn RNA. The fluorescence at the emission maximum (543 nm) was measured as a function of molar fraction [DFHO]/([DFHO]+[Corn RNA]). Mean and standard errors of three independent experiments ( Supplementary Fig. 2b ). The maximum (0.38), indicates 2:1 stoichiometry of RNA to DFHO. ( d ) Kratky analysis of experimental free- and DFHO-bound Corn RNA SAXS data.
Figure Legend Snippet: Biophysical analysis of Corn-DFHO dimer. ( a ) Non-normalized c (s) distributions for Corn RNA at three concentrations and tRNA Lys3 at a single concentration. ( b ) Fluorescence-size exclusion chromatogram (F-SEC) for the Corn-DFHO complex. Absorbance was monitored at 260 nm and fluorescence at the emission maximum (543 nm). Arrow denotes void volume (8 mL) as determined by an independent run with blue dextran (MW ~ 6 MDa) under identical conditions. Elution volume of the 76 nt tRNA Lys3 (10.9 mL) in an independent experiment under identical conditions is also indicated. ( c ) Job plot 23 for DFHO binding to Corn RNA. The fluorescence at the emission maximum (543 nm) was measured as a function of molar fraction [DFHO]/([DFHO]+[Corn RNA]). Mean and standard errors of three independent experiments ( Supplementary Fig. 2b ). The maximum (0.38), indicates 2:1 stoichiometry of RNA to DFHO. ( d ) Kratky analysis of experimental free- and DFHO-bound Corn RNA SAXS data.

Techniques Used: Concentration Assay, Fluorescence, Size-exclusion Chromatography, Multiple Displacement Amplification, Binding Assay

70) Product Images from "FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes"

Article Title: FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18112234

Ultrastructure of islet-like aggregates and insulin secretion assay. PANC-1 cells (70% of confluence) were treated with 50% trypsin for 30 s. and incubated with serum-free medium supplemented with 0.1% BSA plus 1.1 mg/L transferrin (SDT). SDT medium was then supplemented with 500 ng/mL FGF-2b or 500 ng/mL hPL-A, or both hormones (FGF-2b plus hPL-A). After 96 h, aggregates were harvested, fixed and analyzed by electron microscopy. SDT aggregates were composed by acinar cells (Z, zymogen granules; RER, endoplasmic reticulum ( A ). The ultrastructure of cell aggregates obtained with FGF-2b and hPL-A treatment, respectively (N, nucleus; Z, zymogen granules; D, ductal cell) ( B , C ) . Cell aggregates obtained with FGF-2b plus hPL-A treatment showed the pre sence of acinar cells (Z, zymogen granules ( D )) in the border; and a core composed by some: δ cells (δ ( E )); and β cells with: mature (β ( E )); and immature (β ( F )) granules. Insulin secretion assay is shown ( G ): after 96 h of treatment, aggregates were washed and incubated in Krebs/HEPES buffer supplemented with 0.1% BSA for 3 h. Then, glucose 5 mM or 20 mM (or KCl 300 μM) was added for 1 h to stimulate insulin secretion. Glucose-induced insulin secretion was analyzed with insulin-RIA kit and normalized for the total protein content. * p
Figure Legend Snippet: Ultrastructure of islet-like aggregates and insulin secretion assay. PANC-1 cells (70% of confluence) were treated with 50% trypsin for 30 s. and incubated with serum-free medium supplemented with 0.1% BSA plus 1.1 mg/L transferrin (SDT). SDT medium was then supplemented with 500 ng/mL FGF-2b or 500 ng/mL hPL-A, or both hormones (FGF-2b plus hPL-A). After 96 h, aggregates were harvested, fixed and analyzed by electron microscopy. SDT aggregates were composed by acinar cells (Z, zymogen granules; RER, endoplasmic reticulum ( A ). The ultrastructure of cell aggregates obtained with FGF-2b and hPL-A treatment, respectively (N, nucleus; Z, zymogen granules; D, ductal cell) ( B , C ) . Cell aggregates obtained with FGF-2b plus hPL-A treatment showed the pre sence of acinar cells (Z, zymogen granules ( D )) in the border; and a core composed by some: δ cells (δ ( E )); and β cells with: mature (β ( E )); and immature (β ( F )) granules. Insulin secretion assay is shown ( G ): after 96 h of treatment, aggregates were washed and incubated in Krebs/HEPES buffer supplemented with 0.1% BSA for 3 h. Then, glucose 5 mM or 20 mM (or KCl 300 μM) was added for 1 h to stimulate insulin secretion. Glucose-induced insulin secretion was analyzed with insulin-RIA kit and normalized for the total protein content. * p

Techniques Used: Incubation, Electron Microscopy

71) Product Images from "Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody"

Article Title: Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody

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

doi: 10.1073/pnas.1700891114

Analysis of 5-HT 2B /ERG-Fab complex formation using aSEC and a pull-down assay. ( A ) aSEC UV absorption traces of 5 µg of 5-HT 2B /ERG (black), 5 µg Fab (blue), or 5 µg 5-HT 2B /ERG incubated with 5 µg Fab for 1 h on ice (red). Upon binding to 5-HT 2B /ERG, no more free Fab is detected. Complex formation is confirmed according to a nearly doubled peak volume of the receptor species and a shift to shorter retention time indicating an increase in molecular weight ( Inset ). ( B ) SDS/PAGE analysis of pull-down experiments using 1 µg of 5-HT 2B /ERG, 1 µg of His-tagged Fab, or 1 µg of 5-HT 2B /ERG mixed with His-tagged Fab. All samples were incubated on ice for 1 h, bound to TALON resin, and eluted using 20 mM Hepes pH 7.4, 500 mM NaCl, 0.05/0.01% DDM/CHS. Loaded material (Load), flow through (FT), and eluate (E) were analyzed using SDS/PAGE. Analysis confirms 5-HT 2B /ERG-Fab complex formation as His-tagged Fab can capture 5-HT 2B /ERG which otherwise does not bind the resin.
Figure Legend Snippet: Analysis of 5-HT 2B /ERG-Fab complex formation using aSEC and a pull-down assay. ( A ) aSEC UV absorption traces of 5 µg of 5-HT 2B /ERG (black), 5 µg Fab (blue), or 5 µg 5-HT 2B /ERG incubated with 5 µg Fab for 1 h on ice (red). Upon binding to 5-HT 2B /ERG, no more free Fab is detected. Complex formation is confirmed according to a nearly doubled peak volume of the receptor species and a shift to shorter retention time indicating an increase in molecular weight ( Inset ). ( B ) SDS/PAGE analysis of pull-down experiments using 1 µg of 5-HT 2B /ERG, 1 µg of His-tagged Fab, or 1 µg of 5-HT 2B /ERG mixed with His-tagged Fab. All samples were incubated on ice for 1 h, bound to TALON resin, and eluted using 20 mM Hepes pH 7.4, 500 mM NaCl, 0.05/0.01% DDM/CHS. Loaded material (Load), flow through (FT), and eluate (E) were analyzed using SDS/PAGE. Analysis confirms 5-HT 2B /ERG-Fab complex formation as His-tagged Fab can capture 5-HT 2B /ERG which otherwise does not bind the resin.

Techniques Used: Pull Down Assay, Incubation, Binding Assay, Molecular Weight, SDS Page, Flow Cytometry

72) Product Images from "Phenylalanine Hydroxylase from Legionella pneumophila Is a Thermostable Enzyme with a Major Functional Role in Pyomelanin Synthesis"

Article Title: Phenylalanine Hydroxylase from Legionella pneumophila Is a Thermostable Enzyme with a Major Functional Role in Pyomelanin Synthesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0046209

Conformational stability of lpPAH. (A) Far-UV CD spectrum of lpPAH (6 µM in 50 mM Na-phosphate buffer, pH 6.5) at 37°C ( ____ ), at 85°C (––) and at 37°C after heating the sample to 100°C (⋅⋅⋅⋅⋅). [θ], mean residual ellipticity. (B) CD-monitored (at 222 nm) thermal denaturation lpPAH (6 µM in 20 mM Na-Hepes, 200 mM NaCl, pH 7.0) without (•) or with (○) 6 µM Fe(II) (added as ferrous ammonium sulphate) and 6 µM Fe(II) and 5 mM L-Phe (▾). The lines show a fitting of the data to a two-state unfolding equation [79] and points are averaged over ten data points after conversion to fraction unfolded [80] . (C) DSC-monitored thermal denaturation of lpPAH (30 µM) in 20 mM Na-Hepes, pH 7.0. The scan rate was 1°C/min.
Figure Legend Snippet: Conformational stability of lpPAH. (A) Far-UV CD spectrum of lpPAH (6 µM in 50 mM Na-phosphate buffer, pH 6.5) at 37°C ( ____ ), at 85°C (––) and at 37°C after heating the sample to 100°C (⋅⋅⋅⋅⋅). [θ], mean residual ellipticity. (B) CD-monitored (at 222 nm) thermal denaturation lpPAH (6 µM in 20 mM Na-Hepes, 200 mM NaCl, pH 7.0) without (•) or with (○) 6 µM Fe(II) (added as ferrous ammonium sulphate) and 6 µM Fe(II) and 5 mM L-Phe (▾). The lines show a fitting of the data to a two-state unfolding equation [79] and points are averaged over ten data points after conversion to fraction unfolded [80] . (C) DSC-monitored thermal denaturation of lpPAH (30 µM) in 20 mM Na-Hepes, pH 7.0. The scan rate was 1°C/min.

Techniques Used:

73) Product Images from "Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity"

Article Title: Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049048

Kinetic analysis of PI-1 SrtC1 and SrtC2 wild-type and mutants. Triplicate data sets for each experiment were used to calculate the steady-state velocity at different PI-1 peptides concentrations for each enzyme and were expressed as initial rates (µM/s) versus concentration of substrate. SrtC1 (top) and SrtC2 (bottom) enzymes carrying the mutation Y92A and F86A (SrtC1 Y92A and SrtC2 F86A ) and the deletion of the entire N-terminal region (SrtC1 ΔNT and SrtC2 ΔNT ) were analyzed in comparison with wild-type enzymes by FRET assays at various concentrations of three different PI-1 peptides ( Table 2 ). The reactions containing 25 µM of enzyme and 2–128 µM of fluorescent peptide were performed at 37°C in 20 mM HEPES pH 7.5, 100 mM NaCl and 1 mM DTT.
Figure Legend Snippet: Kinetic analysis of PI-1 SrtC1 and SrtC2 wild-type and mutants. Triplicate data sets for each experiment were used to calculate the steady-state velocity at different PI-1 peptides concentrations for each enzyme and were expressed as initial rates (µM/s) versus concentration of substrate. SrtC1 (top) and SrtC2 (bottom) enzymes carrying the mutation Y92A and F86A (SrtC1 Y92A and SrtC2 F86A ) and the deletion of the entire N-terminal region (SrtC1 ΔNT and SrtC2 ΔNT ) were analyzed in comparison with wild-type enzymes by FRET assays at various concentrations of three different PI-1 peptides ( Table 2 ). The reactions containing 25 µM of enzyme and 2–128 µM of fluorescent peptide were performed at 37°C in 20 mM HEPES pH 7.5, 100 mM NaCl and 1 mM DTT.

Techniques Used: Concentration Assay, Mutagenesis

FRET assay with PI-1 and PI-2a peptides for substrate specificity analysis of PI-1 SrtC1 and SrtC2. (A) The reaction solutions contained 128 µM of PI-1 fluorescent peptides and 25 µM of SrtC1-TM (left panel) or SrtC2-TM (right panel). The reactions were performed at 37°C in the assay buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl and 1 mM DTT. Fluorescence emission was monitored every 10 minutes and an increase in fluorescence intensity was observed in the presence of BP, AP1 and AP2 peptides mimicking the LPXTG motif of PI-1 pilus proteins. (B) Reactions were performed with PI-2a peptides and 25 µM of SrtC1-TM in the same conditions described above. (C) In vivo substrate specificity analysis. Immunoblots of total protein extracts from GBS 515 (containing SrtC1 and SrtC2 of pilus island 2a) and JM9130013 (containing SrtC1 and SrtC2 of pilus islands 1 and 2b) wild-type and complemented strains with plasmids expressing the backbone proteins of PI-1 (BP-1) and PI-2a (BP-2a), respectively. The nitrocellulose membranes were probed with antisera specific for BP-1 and BP-2a.
Figure Legend Snippet: FRET assay with PI-1 and PI-2a peptides for substrate specificity analysis of PI-1 SrtC1 and SrtC2. (A) The reaction solutions contained 128 µM of PI-1 fluorescent peptides and 25 µM of SrtC1-TM (left panel) or SrtC2-TM (right panel). The reactions were performed at 37°C in the assay buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl and 1 mM DTT. Fluorescence emission was monitored every 10 minutes and an increase in fluorescence intensity was observed in the presence of BP, AP1 and AP2 peptides mimicking the LPXTG motif of PI-1 pilus proteins. (B) Reactions were performed with PI-2a peptides and 25 µM of SrtC1-TM in the same conditions described above. (C) In vivo substrate specificity analysis. Immunoblots of total protein extracts from GBS 515 (containing SrtC1 and SrtC2 of pilus island 2a) and JM9130013 (containing SrtC1 and SrtC2 of pilus islands 1 and 2b) wild-type and complemented strains with plasmids expressing the backbone proteins of PI-1 (BP-1) and PI-2a (BP-2a), respectively. The nitrocellulose membranes were probed with antisera specific for BP-1 and BP-2a.

Techniques Used: Fluorescence, In Vivo, Western Blot, Expressing

74) Product Images from "p27Kip1 Inhibits Cyclin D-Cyclin-Dependent Kinase 4 by Two Independent Modes ▿"

Article Title: p27Kip1 Inhibits Cyclin D-Cyclin-Dependent Kinase 4 by Two Independent Modes ▿

Journal:

doi: 10.1128/MCB.00898-08

p27-associated complexes from G 0 cells are resistant to exogenous CAK phosphorylation in vitro while those isolated from A cells can be phosphorylated. (A) Lysates were immunoprecipitated (Ip) with p27 antibodies and treated with exogenous cyclin H-cdk7
Figure Legend Snippet: p27-associated complexes from G 0 cells are resistant to exogenous CAK phosphorylation in vitro while those isolated from A cells can be phosphorylated. (A) Lysates were immunoprecipitated (Ip) with p27 antibodies and treated with exogenous cyclin H-cdk7

Techniques Used: In Vitro, Isolation, Immunoprecipitation

CAK is present in G 0 cells and able to phosphorylate recombinant cyclin D-cdk4 in vitro. (A) A and G 0 lysates were directly immunoblotted with cdk7 antibodies (W). Actin was used as a loading control. (B) A and G 0 lysates were immunoprecipitated with
Figure Legend Snippet: CAK is present in G 0 cells and able to phosphorylate recombinant cyclin D-cdk4 in vitro. (A) A and G 0 lysates were directly immunoblotted with cdk7 antibodies (W). Actin was used as a loading control. (B) A and G 0 lysates were immunoprecipitated with

Techniques Used: Recombinant, In Vitro, Immunoprecipitation

75) Product Images from "Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep-2"

Article Title: Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep-2

Journal: British Journal of Pharmacology

doi: 10.1038/bjp.2008.260

HPLC analysis of ANG1005 in formulation. Representative chromatograms of ANG1005 solubilized in dimethyl sulphoxide (DMSO) 100% and ANG1005 in Solutol/Ringer's HEPES buffer formulation. ANG1005 was solubilized in DMSO 100% or formulated as described in the Methods section. Both preparations were analysed by HPLC.
Figure Legend Snippet: HPLC analysis of ANG1005 in formulation. Representative chromatograms of ANG1005 solubilized in dimethyl sulphoxide (DMSO) 100% and ANG1005 in Solutol/Ringer's HEPES buffer formulation. ANG1005 was solubilized in DMSO 100% or formulated as described in the Methods section. Both preparations were analysed by HPLC.

Techniques Used: High Performance Liquid Chromatography

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Transduction:

Article Title: VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase
Article Snippet: .. The lysates were incubated with anti-Flag beads (Anti-Flag M2 Affinity Gel, A2220, Sigma) or anti-VAPB (R2986, Division of Signal Transduction Therapy Unit (DSTT) at the University of Dundee) antibodies cross-linked to Protein A-Sepharose Fast Flow beads (PAS beads, GE Healthcare Life Sciences, Little Chalfont, UK). ..

Centrifugation:

Article Title: Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞
Article Snippet: Crude mitochondrial membranes were suspended in 0.5 ml of isolation medium (250 mM mannitol, 5 mM HEPES/KOH pH 7.4, and 0.5 mM EGTA/KOH), layered on Percoll solution [225 mM mannitol, 25 mM HEPES/KOH, pH 7.4, 1 mM EGTA/KOH, and 30% (v/v) Percoll (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK)], and centrifuged at 95,000 g for 30 min in an SW 55Ti rotor. .. Purified mitochondrial and MAM fractions were collected and washed twice with an isolation medium by centrifugation.

Filtration:

Article Title: Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain
Article Snippet: .. The molecular weight of the CBS Zn-ribbon-like proteins in solution was estimated using a Superdex 75 10/300 GL gel filtration column (Amersham Biosciences) equilibrated with 50 mM Na+ -Hepes (pH 7.5), 0.2M NaCl, and 0.5 mM Tris-2-carboxyethylphosphine-HCl (TCEP), attached to an AKTA FPLC system (Amersham Biosciences). ..

Incubation:

Article Title: VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase
Article Snippet: .. The lysates were incubated with anti-Flag beads (Anti-Flag M2 Affinity Gel, A2220, Sigma) or anti-VAPB (R2986, Division of Signal Transduction Therapy Unit (DSTT) at the University of Dundee) antibodies cross-linked to Protein A-Sepharose Fast Flow beads (PAS beads, GE Healthcare Life Sciences, Little Chalfont, UK). ..

Article Title: Replication of 2-hydroxyadenine-containing DNA and recognition by human MutS?
Article Snippet: After 3min the dNTP (50μM) were added and incubated at 55 °C for 15 min. .. The subsequent elongation was performed by adding 50μM of dNTPs for further 15 min. Fluorescent bands were visualized by Typhoon 9200 Gel Imager (Amersham Bio-sciences Europe GmbH) and quantitated by ImageQuant TL software.

Expressing:

Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation
Article Snippet: Cells and antibodies Wild type and mutant viruses expressing HEK-293T cells were cultured in DMEM with 5% bovine growth serum (Gibco, Carlsbad, CA). .. PBMCs were maintained in RPMI with 10% fetal bovine serum (FBS) (Hyclone, South Logan, Utah).

Modification:

Article Title: Fluopsin C induces oncosis of human breast adenocarcinoma cells
Article Snippet: .. The MCF-7 and HL7702 cells were maintained in RPMI-1640 medium (Hyclone, Thermo Fisher Scientific, Beijing, China), and the MD-MBA-231 cells were cultured in modified DMEM (Gibco, Invitrogen Corporation, Grand Island, NY, USA). ..

Derivative Assay:

Article Title: Fluopsin C induces oncosis of human breast adenocarcinoma cells
Article Snippet: Another normal cell line, HMLE, which was derived from normal human mammary epithelial cells immortalized with the catalytic subunit of telomerase and SV40 large-T and small-T antigens, was kindly provided by Dr Chang-jun ZHU (Shandong University). .. The MCF-7 and HL7702 cells were maintained in RPMI-1640 medium (Hyclone, Thermo Fisher Scientific, Beijing, China), and the MD-MBA-231 cells were cultured in modified DMEM (Gibco, Invitrogen Corporation, Grand Island, NY, USA).

Flow Cytometry:

Article Title: VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase
Article Snippet: .. The lysates were incubated with anti-Flag beads (Anti-Flag M2 Affinity Gel, A2220, Sigma) or anti-VAPB (R2986, Division of Signal Transduction Therapy Unit (DSTT) at the University of Dundee) antibodies cross-linked to Protein A-Sepharose Fast Flow beads (PAS beads, GE Healthcare Life Sciences, Little Chalfont, UK). ..

Immunoprecipitation:

Article Title: VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase
Article Snippet: Paragraph title: Cell extracts and immunoprecipitation ... The lysates were incubated with anti-Flag beads (Anti-Flag M2 Affinity Gel, A2220, Sigma) or anti-VAPB (R2986, Division of Signal Transduction Therapy Unit (DSTT) at the University of Dundee) antibodies cross-linked to Protein A-Sepharose Fast Flow beads (PAS beads, GE Healthcare Life Sciences, Little Chalfont, UK).

Protease Inhibitor:

Article Title: VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase
Article Snippet: Cell extracts and immunoprecipitation For immunoprecipitation, the cells were lysed in buffer A (50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/KOH, pH 7.2; 5 mM Mg(OAc)2 ; 70 mM KOAc; 0.2% Triton X-100; 10% glycerol; 0.2 mM ethylenediaminetetraacetic acid (EDTA); complete protease inhibitor cocktail (Roche, Mannheim, Germany). .. The lysates were incubated with anti-Flag beads (Anti-Flag M2 Affinity Gel, A2220, Sigma) or anti-VAPB (R2986, Division of Signal Transduction Therapy Unit (DSTT) at the University of Dundee) antibodies cross-linked to Protein A-Sepharose Fast Flow beads (PAS beads, GE Healthcare Life Sciences, Little Chalfont, UK).

Cell Culture:

Article Title: Resveratrol inhibits the hedgehog signaling pathway and epithelial-mesenchymal transition and suppresses gastric cancer invasion and metastasis
Article Snippet: Paragraph title: Reagents and cell culture ... Stock solutions (100 mmol/l resveratrol and 20 mmol/l cyclopamine in DMSO) were stored in the dark at −20°C and diluted with RPMI 1640 medium (HyClone, Logan, UT, USA) immediately prior to use.

Article Title: Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2
Article Snippet: .. Isolated HSCs were cultured in RPMI 1640 medium (HyClone, Logan, UT, U.S.) supplemented with 10% heat-inactivated FBS (Gibco, BRL Co. Ltd.), 100 U/mL penicillin, and 100 mg/mL streptomycin in 5% CO2 /95% air at 37°C. ..

Article Title: Retinoid-Dependent Restriction of Human Immunodeficiency Virus Type 1 Replication in Monocytes/Macrophages
Article Snippet: .. U1 cells were cultured in RPMI 1640 medium supplemented with 100 U of penicillin/ml, 100 μg of streptomycin/ml, 0.29 mg of l- glutamine/ml, and 10% fetal bovine serum (FBS) (HyClone, Logan, Utah). .. To prepare monocyte-derived macrophages, CD14+ monocytes, purified from peripheral blood mononuclear cells of normal human donors by using anti-CD14-coated magnetic beads and the auto-MACS protocol (Miltenyi Biotech, Auburn, Calif.) , were allowed to differentiate in culture in the presence of 10% FBS and 10% normal human serum (Atlanta Biologicals, Norcross, Ga.).

Article Title: Fluopsin C induces oncosis of human breast adenocarcinoma cells
Article Snippet: .. The MCF-7 and HL7702 cells were maintained in RPMI-1640 medium (Hyclone, Thermo Fisher Scientific, Beijing, China), and the MD-MBA-231 cells were cultured in modified DMEM (Gibco, Invitrogen Corporation, Grand Island, NY, USA). ..

Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation
Article Snippet: Cells and antibodies Wild type and mutant viruses expressing HEK-293T cells were cultured in DMEM with 5% bovine growth serum (Gibco, Carlsbad, CA). .. PBMCs were maintained in RPMI with 10% fetal bovine serum (FBS) (Hyclone, South Logan, Utah).

Liquid Chromatography:

Article Title: Biochemical Characterization of the SPATE Members EspPα and EspI
Article Snippet: .. EspPα was purified via liquid chromatography (äkta prime FPLC, GE Healthcare, Uppsala, Sweden) using HiTrap Benzamidine FF columns (GE Healthcare, München, Germany) according to the manufacturer’s instructions. .. For purification of EspI, the precipitate was dissolved in 20 mM Tris (tris(hydroxymethyl)aminomethane) buffer containing 50 mM NaCl (pH 6.5).

other:

Article Title: In vitro DNA synthesis opposite oxazolone and repair of this DNA damage using modified oligonucleotides
Article Snippet: T4 polynucleotide kinase, Kf exo– , [γ-32 P]ATP, dNTPs, NAP-25 Sephadex and MicroSpin G-25 columns were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden).

Injection:

Article Title: Anti-Double-Stranded DNA IgG Participates in Renal Fibrosis through Suppressing the Suppressor of Cytokine Signaling 1 Signals
Article Snippet: For anti-dsDNA IgG binding to KIR peptide, anti-dsDNA IgG (10 nM in MES buffer) was immobilized on a CM sensor chip (GE Healthcare, Port Washington, NY, USA), and then KIR peptide (0–250 nM in HEPES buffer) was run on chip. .. For JAK2 loop peptide binding to KIR peptide, the biotinylated KIR peptide (5 nM) was immobilized on streptavidin-coated sensor chip, followed by injection of JAK2 loop peptide (0–250 nM).

Binding Assay:

Article Title: Anti-Double-Stranded DNA IgG Participates in Renal Fibrosis through Suppressing the Suppressor of Cytokine Signaling 1 Signals
Article Snippet: .. For anti-dsDNA IgG binding to KIR peptide, anti-dsDNA IgG (10 nM in MES buffer) was immobilized on a CM sensor chip (GE Healthcare, Port Washington, NY, USA), and then KIR peptide (0–250 nM in HEPES buffer) was run on chip. .. For JAK2 loop peptide binding to KIR peptide, the biotinylated KIR peptide (5 nM) was immobilized on streptavidin-coated sensor chip, followed by injection of JAK2 loop peptide (0–250 nM).

Molecular Weight:

Article Title: Biochemical Characterization of the SPATE Members EspPα and EspI
Article Snippet: The cultures were centrifuged (6000× g , 30 min, 4 °C), supernatants were passed through 0.2 µm Supor machV bottle-top filters (Nalgene, Rochester, NY, USA), and the supernatant was concentrated 20-fold using Vivaflow 200 PES membrane with 50 kDa molecular weight cut off (Vivascience, Hannover, Germany) and Masterflex easy-load peristaltic pump (Cole Parmer, Vernon Hills, Chicago, IL, USA). .. EspPα was purified via liquid chromatography (äkta prime FPLC, GE Healthcare, Uppsala, Sweden) using HiTrap Benzamidine FF columns (GE Healthcare, München, Germany) according to the manufacturer’s instructions.

Article Title: Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain
Article Snippet: .. The molecular weight of the CBS Zn-ribbon-like proteins in solution was estimated using a Superdex 75 10/300 GL gel filtration column (Amersham Biosciences) equilibrated with 50 mM Na+ -Hepes (pH 7.5), 0.2M NaCl, and 0.5 mM Tris-2-carboxyethylphosphine-HCl (TCEP), attached to an AKTA FPLC system (Amersham Biosciences). ..

MTT Assay:

Article Title: Pro-inflammatory effects of a litchi protein extract in murine RAW264.7 macrophages
Article Snippet: Materials and chemicals LPS and thiazolyl blue (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. RPMI-1640 medium, fetal bovine serum and penicillin/streptomycin solution were from Hyclone.

In Vivo:

Article Title: Development of a Curative Therapeutic Vaccine (TheraVac) for the Treatment of Large Established Tumors
Article Snippet: The morphology, in vitro and in vivo growth rate and metastatic ability of cell lines were routinely monitored for stability and consistency. .. All cell lines were maintained in RPMI-1640 medium [RPMI-1640 (Meditech) supplemented with 10% FBS (Hyclone) and 2 mmol/L glutamine, 25 mmol/L HEPES, 100 U/ml penicillin, 100 ug/ml streptomycin, and 50 umol/L 2-mercaptoethanol] at 37 °C in a humidified incubator with 5% CO2 .

Magnetic Beads:

Article Title: Retinoid-Dependent Restriction of Human Immunodeficiency Virus Type 1 Replication in Monocytes/Macrophages
Article Snippet: U1 cells were cultured in RPMI 1640 medium supplemented with 100 U of penicillin/ml, 100 μg of streptomycin/ml, 0.29 mg of l- glutamine/ml, and 10% fetal bovine serum (FBS) (HyClone, Logan, Utah). .. To prepare monocyte-derived macrophages, CD14+ monocytes, purified from peripheral blood mononuclear cells of normal human donors by using anti-CD14-coated magnetic beads and the auto-MACS protocol (Miltenyi Biotech, Auburn, Calif.) , were allowed to differentiate in culture in the presence of 10% FBS and 10% normal human serum (Atlanta Biologicals, Norcross, Ga.).

Mutagenesis:

Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation
Article Snippet: Cells and antibodies Wild type and mutant viruses expressing HEK-293T cells were cultured in DMEM with 5% bovine growth serum (Gibco, Carlsbad, CA). .. PBMCs were maintained in RPMI with 10% fetal bovine serum (FBS) (Hyclone, South Logan, Utah).

Isolation:

Article Title: Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2
Article Snippet: .. Isolated HSCs were cultured in RPMI 1640 medium (HyClone, Logan, UT, U.S.) supplemented with 10% heat-inactivated FBS (Gibco, BRL Co. Ltd.), 100 U/mL penicillin, and 100 mg/mL streptomycin in 5% CO2 /95% air at 37°C. ..

Article Title: Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞
Article Snippet: .. Crude mitochondrial membranes were suspended in 0.5 ml of isolation medium (250 mM mannitol, 5 mM HEPES/KOH pH 7.4, and 0.5 mM EGTA/KOH), layered on Percoll solution [225 mM mannitol, 25 mM HEPES/KOH, pH 7.4, 1 mM EGTA/KOH, and 30% (v/v) Percoll (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK)], and centrifuged at 95,000 g for 30 min in an SW 55Ti rotor. .. Purified mitochondrial and MAM fractions were collected and washed twice with an isolation medium by centrifugation.

Purification:

Article Title: Biochemical Characterization of the SPATE Members EspPα and EspI
Article Snippet: .. EspPα was purified via liquid chromatography (äkta prime FPLC, GE Healthcare, Uppsala, Sweden) using HiTrap Benzamidine FF columns (GE Healthcare, München, Germany) according to the manufacturer’s instructions. .. For purification of EspI, the precipitate was dissolved in 20 mM Tris (tris(hydroxymethyl)aminomethane) buffer containing 50 mM NaCl (pH 6.5).

Article Title: Retinoid-Dependent Restriction of Human Immunodeficiency Virus Type 1 Replication in Monocytes/Macrophages
Article Snippet: U1 cells were cultured in RPMI 1640 medium supplemented with 100 U of penicillin/ml, 100 μg of streptomycin/ml, 0.29 mg of l- glutamine/ml, and 10% fetal bovine serum (FBS) (HyClone, Logan, Utah). .. To prepare monocyte-derived macrophages, CD14+ monocytes, purified from peripheral blood mononuclear cells of normal human donors by using anti-CD14-coated magnetic beads and the auto-MACS protocol (Miltenyi Biotech, Auburn, Calif.) , were allowed to differentiate in culture in the presence of 10% FBS and 10% normal human serum (Atlanta Biologicals, Norcross, Ga.).

Article Title: Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞
Article Snippet: Crude mitochondrial membranes were suspended in 0.5 ml of isolation medium (250 mM mannitol, 5 mM HEPES/KOH pH 7.4, and 0.5 mM EGTA/KOH), layered on Percoll solution [225 mM mannitol, 25 mM HEPES/KOH, pH 7.4, 1 mM EGTA/KOH, and 30% (v/v) Percoll (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK)], and centrifuged at 95,000 g for 30 min in an SW 55Ti rotor. .. Purified mitochondrial and MAM fractions were collected and washed twice with an isolation medium by centrifugation.

Article Title: Replication of 2-hydroxyadenine-containing DNA and recognition by human MutS?
Article Snippet: Primer/template (50 nM) were pre-incubated with 150nM DNA polymerase 4 (Dpo4), purified as described in Gruz et al. [ ], at 55 °C in a buffer containing 30mM potassium phosphate, pH 7.4, 7.5mM MgCl2 , 1.25mM β-mercaptoethanol and 5% glycerol. .. The subsequent elongation was performed by adding 50μM of dNTPs for further 15 min. Fluorescent bands were visualized by Typhoon 9200 Gel Imager (Amersham Bio-sciences Europe GmbH) and quantitated by ImageQuant TL software.

Fast Protein Liquid Chromatography:

Article Title: Biochemical Characterization of the SPATE Members EspPα and EspI
Article Snippet: .. EspPα was purified via liquid chromatography (äkta prime FPLC, GE Healthcare, Uppsala, Sweden) using HiTrap Benzamidine FF columns (GE Healthcare, München, Germany) according to the manufacturer’s instructions. .. For purification of EspI, the precipitate was dissolved in 20 mM Tris (tris(hydroxymethyl)aminomethane) buffer containing 50 mM NaCl (pH 6.5).

Article Title: Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain
Article Snippet: .. The molecular weight of the CBS Zn-ribbon-like proteins in solution was estimated using a Superdex 75 10/300 GL gel filtration column (Amersham Biosciences) equilibrated with 50 mM Na+ -Hepes (pH 7.5), 0.2M NaCl, and 0.5 mM Tris-2-carboxyethylphosphine-HCl (TCEP), attached to an AKTA FPLC system (Amersham Biosciences). ..

Mouse Assay:

Article Title: Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2
Article Snippet: Adult male BALB/c mice (H-2d, haplotype, 8-12 weeks-of-age) mice were purchased from the National Rodent Laboratory Animal Resources, Shanghai, China. .. The mouse H22 hepatoma cell line was purchased from Shanghai Cell Bank, Chinese Academy of Sciences, and maintained in RPMI 1640 medium (HyClone, Logan, UT), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, as previously described [ ].

Article Title: Development of a Curative Therapeutic Vaccine (TheraVac) for the Treatment of Large Established Tumors
Article Snippet: Paragraph title: Mice and cell lines ... All cell lines were maintained in RPMI-1640 medium [RPMI-1640 (Meditech) supplemented with 10% FBS (Hyclone) and 2 mmol/L glutamine, 25 mmol/L HEPES, 100 U/ml penicillin, 100 ug/ml streptomycin, and 50 umol/L 2-mercaptoethanol] at 37 °C in a humidified incubator with 5% CO2 .

Chromatin Immunoprecipitation:

Article Title: Anti-Double-Stranded DNA IgG Participates in Renal Fibrosis through Suppressing the Suppressor of Cytokine Signaling 1 Signals
Article Snippet: .. For anti-dsDNA IgG binding to KIR peptide, anti-dsDNA IgG (10 nM in MES buffer) was immobilized on a CM sensor chip (GE Healthcare, Port Washington, NY, USA), and then KIR peptide (0–250 nM in HEPES buffer) was run on chip. .. For JAK2 loop peptide binding to KIR peptide, the biotinylated KIR peptide (5 nM) was immobilized on streptavidin-coated sensor chip, followed by injection of JAK2 loop peptide (0–250 nM).

SPR Assay:

Article Title: Anti-Double-Stranded DNA IgG Participates in Renal Fibrosis through Suppressing the Suppressor of Cytokine Signaling 1 Signals
Article Snippet: Paragraph title: Surface Plasmon Resonance ... For anti-dsDNA IgG binding to KIR peptide, anti-dsDNA IgG (10 nM in MES buffer) was immobilized on a CM sensor chip (GE Healthcare, Port Washington, NY, USA), and then KIR peptide (0–250 nM in HEPES buffer) was run on chip.

Software:

Article Title: Replication of 2-hydroxyadenine-containing DNA and recognition by human MutS?
Article Snippet: .. The subsequent elongation was performed by adding 50μM of dNTPs for further 15 min. Fluorescent bands were visualized by Typhoon 9200 Gel Imager (Amersham Bio-sciences Europe GmbH) and quantitated by ImageQuant TL software. ..

In Vitro:

Article Title: Development of a Curative Therapeutic Vaccine (TheraVac) for the Treatment of Large Established Tumors
Article Snippet: The morphology, in vitro and in vivo growth rate and metastatic ability of cell lines were routinely monitored for stability and consistency. .. All cell lines were maintained in RPMI-1640 medium [RPMI-1640 (Meditech) supplemented with 10% FBS (Hyclone) and 2 mmol/L glutamine, 25 mmol/L HEPES, 100 U/ml penicillin, 100 ug/ml streptomycin, and 50 umol/L 2-mercaptoethanol] at 37 °C in a humidified incubator with 5% CO2 .

Homogenization:

Article Title: Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞
Article Snippet: Harvested cells were homogenized by a glass Dounce homogenizer in a homogenization buffer (0.25 M sucrose and 10 mM HEPES/KOH, pH 7.4). .. Crude mitochondrial membranes were suspended in 0.5 ml of isolation medium (250 mM mannitol, 5 mM HEPES/KOH pH 7.4, and 0.5 mM EGTA/KOH), layered on Percoll solution [225 mM mannitol, 25 mM HEPES/KOH, pH 7.4, 1 mM EGTA/KOH, and 30% (v/v) Percoll (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK)], and centrifuged at 95,000 g for 30 min in an SW 55Ti rotor.

Quantitation Assay:

Article Title: Anti-Double-Stranded DNA IgG Participates in Renal Fibrosis through Suppressing the Suppressor of Cytokine Signaling 1 Signals
Article Snippet: Surface Plasmon Resonance The Biacore 3000 instrument (Biacore, Piscataway, NJ, USA) was used for the quantitation of binding affinities ( , ). .. For anti-dsDNA IgG binding to KIR peptide, anti-dsDNA IgG (10 nM in MES buffer) was immobilized on a CM sensor chip (GE Healthcare, Port Washington, NY, USA), and then KIR peptide (0–250 nM in HEPES buffer) was run on chip.

Concentration Assay:

Article Title: Resveratrol inhibits the hedgehog signaling pathway and epithelial-mesenchymal transition and suppresses gastric cancer invasion and metastasis
Article Snippet: Stock solutions (100 mmol/l resveratrol and 20 mmol/l cyclopamine in DMSO) were stored in the dark at −20°C and diluted with RPMI 1640 medium (HyClone, Logan, UT, USA) immediately prior to use. .. The final concentration of DMSO in the RPMI 1640 medium was maintained at < 0.1%.

Fractionation:

Article Title: Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞
Article Snippet: Paragraph title: MAM Fractionation. ... Crude mitochondrial membranes were suspended in 0.5 ml of isolation medium (250 mM mannitol, 5 mM HEPES/KOH pH 7.4, and 0.5 mM EGTA/KOH), layered on Percoll solution [225 mM mannitol, 25 mM HEPES/KOH, pH 7.4, 1 mM EGTA/KOH, and 30% (v/v) Percoll (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK)], and centrifuged at 95,000 g for 30 min in an SW 55Ti rotor.

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  • 99
    GE Healthcare percoll solution
    MAM accommodates DRMs associated with Sig-1R. A, purification of MAM and microsomal fractions from CHO cells. Nuclear (P1), mitochondrial (Mito), MAM, microsomal (P3), and cytosolic (Cyt) fractions were prepared by differential centrifugation combined with a <t>Percoll</t> gradient fractionation. Five micrograms of proteins was applied to SDS-PAGE, followed by immunoblotting. COX, cytochrome c oxidase subunit I. Asterisks indicate ER chaperones involved in vesicular transport. Graphs represent fraction distributions of proteins in which the sum of five fractions was taken as 100% for each protein. B, Sig-1R-associated DRMs in the MAM. MAM and microsomes (P3) (25 μg of total proteins in each) were extracted by 0.5% Tx (Tx-100) or Triton X-114 at 4°C. DRMs and detergent-soluble supernatant (S) were prepared by differential centrifugations. The numbers represent the average of optical density (O.D.) measured in each protein band ( n = 3). C, silver staining for total proteins associated with DRMs in MAM and microsomal fractions. MAM and microsomes (25 μg of total proteins in each) were extracted in 0.5% Tx at 4°C, and DRMs and soluble supernatants (S) were prepared. Proteins were visualized by 13% SDS-PAGE, followed by silver staining. The numbers represent the average of O.D. measured in each lane ( n = 3). MW, molecular weight of standard proteins. D, lipid contents in MAM and microsomal fractions. Lipids were extracted and analyzed by HPTLC. Cholesterol (Chol) was detected using a ferric chloride spray; GlcCer using a diphenylamine-aniline spray. Lipids in the second panel were visualized under UV light after an ANS spray. In the lipid overlay assay for ceramides (Cer, bottom), ceramides extracted from HPTLC plates were immobilized on a nitrocellulose membrane followed by immunoblotting with anti-ceramide antibodies. SM, sphingomyelin.
    Percoll Solution, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare hepes
    In vitro fibril formation of sumoylated α-synuclein. (A) Coomassie staining of α-synuclein sumoylated to 100% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (B) Fibrillization kinetics of α-synuclein sumoylated to 100% (70 µM; closed circles) and control condition (70 µM of nonmodified α-synuclein + 70 µM of free SUMO1; open circles). (C) Nonamyloidogenic amorphous oligomers formed by α-synuclein sumoylated to 100%. (D) Mature fibrils formed by control nonmodified α-synuclein in the presence of 70 µM of free SUMO1. (E) Coomassie staining of α-synuclein sumoylated to 50% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (F) Fibrillization kinetics of α-synuclein sumoylated to 50% (35 µM sumoylated α-synuclein + 35 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 35 µM of free SUMO1; open circles). (G) Nonamyloidogenic amorphous material formed by α-synuclein sumoylated to 50%. (H) Mature fibrils formed by control nonmodified α-synuclein in the presence of 35 µM of free SUMO1. (I) Coomassie staining of α-synuclein sumoylated to 10% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (J) Fibrillization kinetics of α-synuclein sumoylated to 10% (7 µM sumoylated α-synuclein + 63 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 7 µM of free SUMO1; open circles). (K) Mature fibrils formed by α-synuclein sumoylated to 10%. (L) Mature fibrils formed by control nonmodified α-synuclein in the presence of 7 µM of free SUMO1. (C, D, G, H, K, and L) TEM of aggregation samples after 146 h of incubation in 50 mM <t>Hepes</t> and 100 mM <t>NaCl,</t> pH 7.4, at 37°C with constant stirring. ThT, Thioflavin T. Bars, 200 nm.
    Hepes, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare n2a cells
    gSAP regulates Aβ production but does not influence Notch cleavage a: siRNA-mediated knockdown of gSAP in <t>N2a</t> cells overexpressing APP695 lowered Aβ production. The Aβ-lowering effects of imatinib and of siRNA were not additive (mean ±s.d.; ** p
    N2a Cells, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GE Healthcare cdc20 lysis buffer
    Conformational changes of the APC/C between the inactive apo and the active ternary states and domain and sequence analysis of <t>Cdc20.</t> a, b , Subunits that undergo conformational changes upon coactivator and substrate binding are highlighted in their ternary state and coloured as in , while the corresponding proteins in the inactive apo state are in lighter shades. In the active conformation, the platform subdomain containing subunits Apc1, Apc4 and Apc5 is shifted upward, inducing a large movement of the catalytic module to enable E2 access. c , Domain organization of Cdc20. d , Sequence alignment of Cdc20 NTD and Cdh1 NTD with α-helices represented as cylinders (purple and grey for Cdc20 NTD and Cdh1 NTD , respectively) underneath the sequences and the C box and KILR/KLLR motif highlighted. Fig. 1
    Cdc20 Lysis Buffer, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MAM accommodates DRMs associated with Sig-1R. A, purification of MAM and microsomal fractions from CHO cells. Nuclear (P1), mitochondrial (Mito), MAM, microsomal (P3), and cytosolic (Cyt) fractions were prepared by differential centrifugation combined with a Percoll gradient fractionation. Five micrograms of proteins was applied to SDS-PAGE, followed by immunoblotting. COX, cytochrome c oxidase subunit I. Asterisks indicate ER chaperones involved in vesicular transport. Graphs represent fraction distributions of proteins in which the sum of five fractions was taken as 100% for each protein. B, Sig-1R-associated DRMs in the MAM. MAM and microsomes (P3) (25 μg of total proteins in each) were extracted by 0.5% Tx (Tx-100) or Triton X-114 at 4°C. DRMs and detergent-soluble supernatant (S) were prepared by differential centrifugations. The numbers represent the average of optical density (O.D.) measured in each protein band ( n = 3). C, silver staining for total proteins associated with DRMs in MAM and microsomal fractions. MAM and microsomes (25 μg of total proteins in each) were extracted in 0.5% Tx at 4°C, and DRMs and soluble supernatants (S) were prepared. Proteins were visualized by 13% SDS-PAGE, followed by silver staining. The numbers represent the average of O.D. measured in each lane ( n = 3). MW, molecular weight of standard proteins. D, lipid contents in MAM and microsomal fractions. Lipids were extracted and analyzed by HPTLC. Cholesterol (Chol) was detected using a ferric chloride spray; GlcCer using a diphenylamine-aniline spray. Lipids in the second panel were visualized under UV light after an ANS spray. In the lipid overlay assay for ceramides (Cer, bottom), ceramides extracted from HPTLC plates were immobilized on a nitrocellulose membrane followed by immunoblotting with anti-ceramide antibodies. SM, sphingomyelin.

    Journal: Molecular Pharmacology

    Article Title: Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞

    doi: 10.1124/mol.109.062539

    Figure Lengend Snippet: MAM accommodates DRMs associated with Sig-1R. A, purification of MAM and microsomal fractions from CHO cells. Nuclear (P1), mitochondrial (Mito), MAM, microsomal (P3), and cytosolic (Cyt) fractions were prepared by differential centrifugation combined with a Percoll gradient fractionation. Five micrograms of proteins was applied to SDS-PAGE, followed by immunoblotting. COX, cytochrome c oxidase subunit I. Asterisks indicate ER chaperones involved in vesicular transport. Graphs represent fraction distributions of proteins in which the sum of five fractions was taken as 100% for each protein. B, Sig-1R-associated DRMs in the MAM. MAM and microsomes (P3) (25 μg of total proteins in each) were extracted by 0.5% Tx (Tx-100) or Triton X-114 at 4°C. DRMs and detergent-soluble supernatant (S) were prepared by differential centrifugations. The numbers represent the average of optical density (O.D.) measured in each protein band ( n = 3). C, silver staining for total proteins associated with DRMs in MAM and microsomal fractions. MAM and microsomes (25 μg of total proteins in each) were extracted in 0.5% Tx at 4°C, and DRMs and soluble supernatants (S) were prepared. Proteins were visualized by 13% SDS-PAGE, followed by silver staining. The numbers represent the average of O.D. measured in each lane ( n = 3). MW, molecular weight of standard proteins. D, lipid contents in MAM and microsomal fractions. Lipids were extracted and analyzed by HPTLC. Cholesterol (Chol) was detected using a ferric chloride spray; GlcCer using a diphenylamine-aniline spray. Lipids in the second panel were visualized under UV light after an ANS spray. In the lipid overlay assay for ceramides (Cer, bottom), ceramides extracted from HPTLC plates were immobilized on a nitrocellulose membrane followed by immunoblotting with anti-ceramide antibodies. SM, sphingomyelin.

    Article Snippet: Crude mitochondrial membranes were suspended in 0.5 ml of isolation medium (250 mM mannitol, 5 mM HEPES/KOH pH 7.4, and 0.5 mM EGTA/KOH), layered on Percoll solution [225 mM mannitol, 25 mM HEPES/KOH, pH 7.4, 1 mM EGTA/KOH, and 30% (v/v) Percoll (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK)], and centrifuged at 95,000 g for 30 min in an SW 55Ti rotor.

    Techniques: Purification, Centrifugation, Fractionation, SDS Page, Silver Staining, Molecular Weight, High Performance Thin Layer Chromatography, Overlay Assay

    In vitro fibril formation of sumoylated α-synuclein. (A) Coomassie staining of α-synuclein sumoylated to 100% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (B) Fibrillization kinetics of α-synuclein sumoylated to 100% (70 µM; closed circles) and control condition (70 µM of nonmodified α-synuclein + 70 µM of free SUMO1; open circles). (C) Nonamyloidogenic amorphous oligomers formed by α-synuclein sumoylated to 100%. (D) Mature fibrils formed by control nonmodified α-synuclein in the presence of 70 µM of free SUMO1. (E) Coomassie staining of α-synuclein sumoylated to 50% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (F) Fibrillization kinetics of α-synuclein sumoylated to 50% (35 µM sumoylated α-synuclein + 35 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 35 µM of free SUMO1; open circles). (G) Nonamyloidogenic amorphous material formed by α-synuclein sumoylated to 50%. (H) Mature fibrils formed by control nonmodified α-synuclein in the presence of 35 µM of free SUMO1. (I) Coomassie staining of α-synuclein sumoylated to 10% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (J) Fibrillization kinetics of α-synuclein sumoylated to 10% (7 µM sumoylated α-synuclein + 63 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 7 µM of free SUMO1; open circles). (K) Mature fibrils formed by α-synuclein sumoylated to 10%. (L) Mature fibrils formed by control nonmodified α-synuclein in the presence of 7 µM of free SUMO1. (C, D, G, H, K, and L) TEM of aggregation samples after 146 h of incubation in 50 mM Hepes and 100 mM NaCl, pH 7.4, at 37°C with constant stirring. ThT, Thioflavin T. Bars, 200 nm.

    Journal: The Journal of Cell Biology

    Article Title: Sumoylation inhibits ?-synuclein aggregation and toxicity

    doi: 10.1083/jcb.201010117

    Figure Lengend Snippet: In vitro fibril formation of sumoylated α-synuclein. (A) Coomassie staining of α-synuclein sumoylated to 100% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (B) Fibrillization kinetics of α-synuclein sumoylated to 100% (70 µM; closed circles) and control condition (70 µM of nonmodified α-synuclein + 70 µM of free SUMO1; open circles). (C) Nonamyloidogenic amorphous oligomers formed by α-synuclein sumoylated to 100%. (D) Mature fibrils formed by control nonmodified α-synuclein in the presence of 70 µM of free SUMO1. (E) Coomassie staining of α-synuclein sumoylated to 50% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (F) Fibrillization kinetics of α-synuclein sumoylated to 50% (35 µM sumoylated α-synuclein + 35 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 35 µM of free SUMO1; open circles). (G) Nonamyloidogenic amorphous material formed by α-synuclein sumoylated to 50%. (H) Mature fibrils formed by control nonmodified α-synuclein in the presence of 35 µM of free SUMO1. (I) Coomassie staining of α-synuclein sumoylated to 10% (lane 1) and α-synuclein with an equimolar amount of free SUMO1 (lane 2). (J) Fibrillization kinetics of α-synuclein sumoylated to 10% (7 µM sumoylated α-synuclein + 63 µM of nonmodified α-synuclein; closed circles) and control condition (70 µM of nonmodified α-synuclein + 7 µM of free SUMO1; open circles). (K) Mature fibrils formed by α-synuclein sumoylated to 10%. (L) Mature fibrils formed by control nonmodified α-synuclein in the presence of 7 µM of free SUMO1. (C, D, G, H, K, and L) TEM of aggregation samples after 146 h of incubation in 50 mM Hepes and 100 mM NaCl, pH 7.4, at 37°C with constant stirring. ThT, Thioflavin T. Bars, 200 nm.

    Article Snippet: After concentration, the sample was further purified using gel filtration in 50 mM Hepes and 100 mM NaCl, pH 7.4 (Superdex S75; GE Healthcare).

    Techniques: In Vitro, Staining, Transmission Electron Microscopy, Incubation

    gSAP regulates Aβ production but does not influence Notch cleavage a: siRNA-mediated knockdown of gSAP in N2a cells overexpressing APP695 lowered Aβ production. The Aβ-lowering effects of imatinib and of siRNA were not additive (mean ±s.d.; ** p

    Journal: Nature

    Article Title: Gamma-secretase activating protein, a therapeutic target for Alzheimer's disease

    doi: 10.1038/nature09325

    Figure Lengend Snippet: gSAP regulates Aβ production but does not influence Notch cleavage a: siRNA-mediated knockdown of gSAP in N2a cells overexpressing APP695 lowered Aβ production. The Aβ-lowering effects of imatinib and of siRNA were not additive (mean ±s.d.; ** p

    Article Snippet: Gel filtration chromatography Solubilized membrane preparations from N2a cells (0.2 ml, ~1 mg of solubilized protein, in 50 mM Hepes, 150 mM NaCl, 1% CHAPSO, 5 mM MgCl2 , 5 mM CaCl2 ) were applied to a Superdex 200 10/300 GL column (GE healthcare) of an AKTA fast performance liquid chromatography system.

    Techniques:

    Identification of gSAP as an imatinib target a: A PS1-associated 16 kDa protein is labeled by a photoactivatable imatinib derivative. Left panel: photolysis of 125 I-G01 with membrane preparations. Middle panel: photolysis of 3 H-G01 with intact HEK293 cells. Right panel: PS1-CTF migrated with a slower mobility than the labeled 16 kDa band and was not labeled by G01. Labeling specificity was confirmed by competition with unlabeled imatinib. b: Solubilized endogenous γ-secretase components from HEK293 cells were bound to immobilized biotin-imatinib (left panel). Among the proteins bound to biotin-imatinib, a ~ 16 kDa band was detected by silver staining and was identified as the C-terminal domain of gSAP (right panel, arrow and label “gSAP”). Biotin-coated beads and an inactive biotin-imatinib derivative (see supplementary Fig. 3 ) served as controls. c: Endogenous gSAP in N2a cells was synthesized as a full length 98 kDa-precursor protein and rapidly processed into a 16 kDa C-terminal fragment. Under steady-state conditions, the predominant cellular form of gSAP was 16 kDa. d: Endogenous gSAP-16K was specifically labeled by 3 H-G01 in neuroblastoma cells. e: After gSAP siRNA knockdown in N2a cells, immobilized biotin-imatinib no longer captured PS1.

    Journal: Nature

    Article Title: Gamma-secretase activating protein, a therapeutic target for Alzheimer's disease

    doi: 10.1038/nature09325

    Figure Lengend Snippet: Identification of gSAP as an imatinib target a: A PS1-associated 16 kDa protein is labeled by a photoactivatable imatinib derivative. Left panel: photolysis of 125 I-G01 with membrane preparations. Middle panel: photolysis of 3 H-G01 with intact HEK293 cells. Right panel: PS1-CTF migrated with a slower mobility than the labeled 16 kDa band and was not labeled by G01. Labeling specificity was confirmed by competition with unlabeled imatinib. b: Solubilized endogenous γ-secretase components from HEK293 cells were bound to immobilized biotin-imatinib (left panel). Among the proteins bound to biotin-imatinib, a ~ 16 kDa band was detected by silver staining and was identified as the C-terminal domain of gSAP (right panel, arrow and label “gSAP”). Biotin-coated beads and an inactive biotin-imatinib derivative (see supplementary Fig. 3 ) served as controls. c: Endogenous gSAP in N2a cells was synthesized as a full length 98 kDa-precursor protein and rapidly processed into a 16 kDa C-terminal fragment. Under steady-state conditions, the predominant cellular form of gSAP was 16 kDa. d: Endogenous gSAP-16K was specifically labeled by 3 H-G01 in neuroblastoma cells. e: After gSAP siRNA knockdown in N2a cells, immobilized biotin-imatinib no longer captured PS1.

    Article Snippet: Gel filtration chromatography Solubilized membrane preparations from N2a cells (0.2 ml, ~1 mg of solubilized protein, in 50 mM Hepes, 150 mM NaCl, 1% CHAPSO, 5 mM MgCl2 , 5 mM CaCl2 ) were applied to a Superdex 200 10/300 GL column (GE healthcare) of an AKTA fast performance liquid chromatography system.

    Techniques: Labeling, Silver Staining, Synthesized

    gSAP interacts with γ-secretase and APP-CTF but not with Notch a: Endogenous gSAP-16K in solubilized membrane preparations from N2a cells co-migrated with γ-secretase components during gel filtration (void volume: fraction 6). b: Immunoprecipitation of endogenous gSAP from N2a cells resulted in co-immunoprecipitation of γ-secretase components. c: Endogenous gSAP-16K and γ-secretase components are highly enriched by an immobilized γ-secretase transition state analogue (GSI beads). d: In HEK293 cells, gSAP-16K and APP-CTF, but not NotchΔE, co-immunoprecipitated. e: Imatinib treatment reduced the co-immunoprecipitation of APP-CTF and gSAP in a concentration-dependent manner. An inactive imatinib derivative (IC200001, see supplementary Fig. 3 ) served as a negative control. f: In HEK293 cells, APP-CTF without the cytoplasmic domain (APPε-CTF) did not co-immunoprecipitate with gSAP-16K (upper panel); γ-cleavage of APPε-CTF was not stimulated by gSAP-16K in an in vitro assay (lower panel).

    Journal: Nature

    Article Title: Gamma-secretase activating protein, a therapeutic target for Alzheimer's disease

    doi: 10.1038/nature09325

    Figure Lengend Snippet: gSAP interacts with γ-secretase and APP-CTF but not with Notch a: Endogenous gSAP-16K in solubilized membrane preparations from N2a cells co-migrated with γ-secretase components during gel filtration (void volume: fraction 6). b: Immunoprecipitation of endogenous gSAP from N2a cells resulted in co-immunoprecipitation of γ-secretase components. c: Endogenous gSAP-16K and γ-secretase components are highly enriched by an immobilized γ-secretase transition state analogue (GSI beads). d: In HEK293 cells, gSAP-16K and APP-CTF, but not NotchΔE, co-immunoprecipitated. e: Imatinib treatment reduced the co-immunoprecipitation of APP-CTF and gSAP in a concentration-dependent manner. An inactive imatinib derivative (IC200001, see supplementary Fig. 3 ) served as a negative control. f: In HEK293 cells, APP-CTF without the cytoplasmic domain (APPε-CTF) did not co-immunoprecipitate with gSAP-16K (upper panel); γ-cleavage of APPε-CTF was not stimulated by gSAP-16K in an in vitro assay (lower panel).

    Article Snippet: Gel filtration chromatography Solubilized membrane preparations from N2a cells (0.2 ml, ~1 mg of solubilized protein, in 50 mM Hepes, 150 mM NaCl, 1% CHAPSO, 5 mM MgCl2 , 5 mM CaCl2 ) were applied to a Superdex 200 10/300 GL column (GE healthcare) of an AKTA fast performance liquid chromatography system.

    Techniques: Filtration, Immunoprecipitation, Concentration Assay, Negative Control, In Vitro

    Conformational changes of the APC/C between the inactive apo and the active ternary states and domain and sequence analysis of Cdc20. a, b , Subunits that undergo conformational changes upon coactivator and substrate binding are highlighted in their ternary state and coloured as in , while the corresponding proteins in the inactive apo state are in lighter shades. In the active conformation, the platform subdomain containing subunits Apc1, Apc4 and Apc5 is shifted upward, inducing a large movement of the catalytic module to enable E2 access. c , Domain organization of Cdc20. d , Sequence alignment of Cdc20 NTD and Cdh1 NTD with α-helices represented as cylinders (purple and grey for Cdc20 NTD and Cdh1 NTD , respectively) underneath the sequences and the C box and KILR/KLLR motif highlighted. Fig. 1

    Journal: Nature

    Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

    doi: 10.1038/nature17973

    Figure Lengend Snippet: Conformational changes of the APC/C between the inactive apo and the active ternary states and domain and sequence analysis of Cdc20. a, b , Subunits that undergo conformational changes upon coactivator and substrate binding are highlighted in their ternary state and coloured as in , while the corresponding proteins in the inactive apo state are in lighter shades. In the active conformation, the platform subdomain containing subunits Apc1, Apc4 and Apc5 is shifted upward, inducing a large movement of the catalytic module to enable E2 access. c , Domain organization of Cdc20. d , Sequence alignment of Cdc20 NTD and Cdh1 NTD with α-helices represented as cylinders (purple and grey for Cdc20 NTD and Cdh1 NTD , respectively) underneath the sequences and the C box and KILR/KLLR motif highlighted. Fig. 1

    Article Snippet: Harvested cell pellets were resuspended in Cdc20 lysis buffer (50 mM Hepes pH 7.8, 500 mM NaCl, 30 mM imidazole, 10% glycerol and 0.5 mM TCEP) supplemented with 0.1 mM PMSF, 5 units/ml benzonase and CompleteTM EDTA-free protease inhibitors and loaded onto a HisTrap HP column (GE Healthcare).

    Techniques: Sequencing, Binding Assay, Cytotoxicity Assay

    Comparison of Cdc20 and Cdh1 association to the APC/C. a , The catalytic module (Apc2-Apc11) of the APC/C Cdc20-Hsl1 complex is flexible and almost no density accounting for Apc11 (pink, modelled based on the structure of APC/C Cdh1-Emi1 could be observed. b , The WD40 domain of Cdc20 (purple) occupies a similar position as Cdh1 WD40 (grey), but it is displaced from the APC/C by as much as 10 Å. c, d , EM density for Cdc20 C box allowed for ab initio model building and the C-box interaction with Apc8B (cyan) is well conserved between the two coactivators. e , Both Cdc20 IR and Cdh1 IR associates with Apc3A (orange), although the EM density for Cdc20 IR is much weaker (not shown) and the C-terminal α-helix in Cdh1 IR is absent.

    Journal: Nature

    Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

    doi: 10.1038/nature17973

    Figure Lengend Snippet: Comparison of Cdc20 and Cdh1 association to the APC/C. a , The catalytic module (Apc2-Apc11) of the APC/C Cdc20-Hsl1 complex is flexible and almost no density accounting for Apc11 (pink, modelled based on the structure of APC/C Cdh1-Emi1 could be observed. b , The WD40 domain of Cdc20 (purple) occupies a similar position as Cdh1 WD40 (grey), but it is displaced from the APC/C by as much as 10 Å. c, d , EM density for Cdc20 C box allowed for ab initio model building and the C-box interaction with Apc8B (cyan) is well conserved between the two coactivators. e , Both Cdc20 IR and Cdh1 IR associates with Apc3A (orange), although the EM density for Cdc20 IR is much weaker (not shown) and the C-terminal α-helix in Cdh1 IR is absent.

    Article Snippet: Harvested cell pellets were resuspended in Cdc20 lysis buffer (50 mM Hepes pH 7.8, 500 mM NaCl, 30 mM imidazole, 10% glycerol and 0.5 mM TCEP) supplemented with 0.1 mM PMSF, 5 units/ml benzonase and CompleteTM EDTA-free protease inhibitors and loaded onto a HisTrap HP column (GE Healthcare).

    Techniques:

    The Apc1 AI segment binds to the C-box binding site and mimics the Cdc20 C box . a , Cdk2-cyclin A2-Cks2 was used for ubiquitination assays. In vitro phosphorylated APC/C (both Cdk2-cyclin A3-Cks2 and Plk1) can be activated by Cdc20 (lanes 1-5). Deletion of the Apc1 300s loop activated the APC/C without phosphorylation (lanes 6-7), and kinase treatment of APC/C ΔApc1-300s does not enhance APC/C activity. The APC/C ΔApc3-loop mutant showed similar activity as unphosphorylated APC/C (lanes 10-11 vs 2-3 and 4-5), but had reduced activation by phosphorylation. Nevertheless, deletion of both Apc1 300s and Apc3 loops (APC/C ΔApc1-300s Apc3-loop ) restored activity to that of WT phosphorylated APC/C and unphosphorylated APC/C ΔApc1-300s (lanes 14-17). b , Identification of the Apc1 AI segment occupying the C-box binding site by assessing the inhibitory effect of eight peptides spanning the Apc1 300s loop. A single peptide (peptide 7, residues 361-380) suppressed the activity of APC/C ΔApc1-300s (lane 9), indicating that this peptide blocks the C-box binding site. A control with WT unphosphorylated APC/C ( unp. APC/C WT ) is in lane 11. c , The Apc1 AI segment (peptide 7, residues 361-380) shares sequence similarity with Cdc20 C box . A model for the AI segment (green) was fitted into the EM density of the apo unphosphorylated APC/C map (grey). Arg368 overlaps with the crucial Arg78 of Cdc20 C box (purple, right panel). The flanking serines shown to be phosphorylated are highlighted as red spheres. Ser377 is outside the observed EM density. d , Mutation of a single Arg368 residue (APC/C Apc1-R368E ) or mutating its four neighbouring serine residues (Ser364, Ser372, Ser373, Ser377) to glutamates (APC/C Apc1-4S/E ) activated the APC/C without phosphorylation. 30 nM Cdc20 was used for assay in a and 20 nM Cdc20 for assays in b and d . Experiments in a and d were replicated three times and in b for gel source data.

    Journal: Nature

    Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

    doi: 10.1038/nature17973

    Figure Lengend Snippet: The Apc1 AI segment binds to the C-box binding site and mimics the Cdc20 C box . a , Cdk2-cyclin A2-Cks2 was used for ubiquitination assays. In vitro phosphorylated APC/C (both Cdk2-cyclin A3-Cks2 and Plk1) can be activated by Cdc20 (lanes 1-5). Deletion of the Apc1 300s loop activated the APC/C without phosphorylation (lanes 6-7), and kinase treatment of APC/C ΔApc1-300s does not enhance APC/C activity. The APC/C ΔApc3-loop mutant showed similar activity as unphosphorylated APC/C (lanes 10-11 vs 2-3 and 4-5), but had reduced activation by phosphorylation. Nevertheless, deletion of both Apc1 300s and Apc3 loops (APC/C ΔApc1-300s Apc3-loop ) restored activity to that of WT phosphorylated APC/C and unphosphorylated APC/C ΔApc1-300s (lanes 14-17). b , Identification of the Apc1 AI segment occupying the C-box binding site by assessing the inhibitory effect of eight peptides spanning the Apc1 300s loop. A single peptide (peptide 7, residues 361-380) suppressed the activity of APC/C ΔApc1-300s (lane 9), indicating that this peptide blocks the C-box binding site. A control with WT unphosphorylated APC/C ( unp. APC/C WT ) is in lane 11. c , The Apc1 AI segment (peptide 7, residues 361-380) shares sequence similarity with Cdc20 C box . A model for the AI segment (green) was fitted into the EM density of the apo unphosphorylated APC/C map (grey). Arg368 overlaps with the crucial Arg78 of Cdc20 C box (purple, right panel). The flanking serines shown to be phosphorylated are highlighted as red spheres. Ser377 is outside the observed EM density. d , Mutation of a single Arg368 residue (APC/C Apc1-R368E ) or mutating its four neighbouring serine residues (Ser364, Ser372, Ser373, Ser377) to glutamates (APC/C Apc1-4S/E ) activated the APC/C without phosphorylation. 30 nM Cdc20 was used for assay in a and 20 nM Cdc20 for assays in b and d . Experiments in a and d were replicated three times and in b for gel source data.

    Article Snippet: Harvested cell pellets were resuspended in Cdc20 lysis buffer (50 mM Hepes pH 7.8, 500 mM NaCl, 30 mM imidazole, 10% glycerol and 0.5 mM TCEP) supplemented with 0.1 mM PMSF, 5 units/ml benzonase and CompleteTM EDTA-free protease inhibitors and loaded onto a HisTrap HP column (GE Healthcare).

    Techniques: Binding Assay, In Vitro, Activity Assay, Mutagenesis, Activation Assay, Sequencing

    Three-dimensional classification of APC/C Cdc20-Hsl . The initial particles after 2-dimensional classification were divided into six classes by 3-dimensional classification module using RELION. The resultant classes were grouped into four categories: (i) 9.0% in the active ternary state with coactivator and substrate bound; (ii) 11.3% in a hybrid state with coactivator bound, but the APC/C in the inactive conformation; (iii) 71.6% in the inactive apo state; (iv) 8.1% has poorer reconstruction due to some bad particles. The first class in the active ternary state containing 179,660 particles was used for 3-dimensional refinement and movie correction to obtain the final reconstruction at 3.9 Å.

    Journal: Nature

    Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

    doi: 10.1038/nature17973

    Figure Lengend Snippet: Three-dimensional classification of APC/C Cdc20-Hsl . The initial particles after 2-dimensional classification were divided into six classes by 3-dimensional classification module using RELION. The resultant classes were grouped into four categories: (i) 9.0% in the active ternary state with coactivator and substrate bound; (ii) 11.3% in a hybrid state with coactivator bound, but the APC/C in the inactive conformation; (iii) 71.6% in the inactive apo state; (iv) 8.1% has poorer reconstruction due to some bad particles. The first class in the active ternary state containing 179,660 particles was used for 3-dimensional refinement and movie correction to obtain the final reconstruction at 3.9 Å.

    Article Snippet: Harvested cell pellets were resuspended in Cdc20 lysis buffer (50 mM Hepes pH 7.8, 500 mM NaCl, 30 mM imidazole, 10% glycerol and 0.5 mM TCEP) supplemented with 0.1 mM PMSF, 5 units/ml benzonase and CompleteTM EDTA-free protease inhibitors and loaded onto a HisTrap HP column (GE Healthcare).

    Techniques:

    Preparations and EM images of different APC/C samples used for structural studies. a , Recombinant human APC/C was in vitro phosphorylated using Cdk2-cyclin A3, Cdk2-cyclin A3-Cks2 or Plk1 alone or with both Cdk2-cyclin A3-Cks2 and Plk1. The phosphorylated APC/C samples are shown on SDS-PAGE. b , In vitro phosphorylated recombinant human APC/C can be fully activated by Cdc20 to ubiquitylate a native substrate Cdk2-cyclin A2-Cks2 when both kinases were added (lanes 9, 10). Without Cks2 (lanes 3, 4) or with Plk1 alone (lanes 7, 8) no activation of the APC/C could be observed, whereas treating with Cdk2-cyclin A3-Cks2 alone (lanes 5, 6) resulted in its partial activation. A time course was recorded at 15 and 30 min and 20 nM of Cdc20 was used. This experiment was replicated three times. Anti-Apc3 antibodies (BD Bioscience, cat. code: 610454) were used as a loading control. c , Purified APC/C WT and mutant samples with and without kinase treatment (both Cdk2-cyclin A3-Cks2 and Plk1). Upon deletion of the Apc3 loop, no association of the Cdk2-cyclin A3-Cks2 kinase to the APC/C could be observed (lanes 6 and 8). d , Purified APC/C Cdc20-Hsl1 ternary complex on SDS-PAGE. e , A typical cryo-EM micrograph of APC/C Cdc20-Hsl1 representative of 15,582 micrographs. f , Gallery of two-dimensional averages of APC/C Cdc20-Hsl1 showing different views; representative of 100 two-dimensional averages. g for gel source data.

    Journal: Nature

    Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

    doi: 10.1038/nature17973

    Figure Lengend Snippet: Preparations and EM images of different APC/C samples used for structural studies. a , Recombinant human APC/C was in vitro phosphorylated using Cdk2-cyclin A3, Cdk2-cyclin A3-Cks2 or Plk1 alone or with both Cdk2-cyclin A3-Cks2 and Plk1. The phosphorylated APC/C samples are shown on SDS-PAGE. b , In vitro phosphorylated recombinant human APC/C can be fully activated by Cdc20 to ubiquitylate a native substrate Cdk2-cyclin A2-Cks2 when both kinases were added (lanes 9, 10). Without Cks2 (lanes 3, 4) or with Plk1 alone (lanes 7, 8) no activation of the APC/C could be observed, whereas treating with Cdk2-cyclin A3-Cks2 alone (lanes 5, 6) resulted in its partial activation. A time course was recorded at 15 and 30 min and 20 nM of Cdc20 was used. This experiment was replicated three times. Anti-Apc3 antibodies (BD Bioscience, cat. code: 610454) were used as a loading control. c , Purified APC/C WT and mutant samples with and without kinase treatment (both Cdk2-cyclin A3-Cks2 and Plk1). Upon deletion of the Apc3 loop, no association of the Cdk2-cyclin A3-Cks2 kinase to the APC/C could be observed (lanes 6 and 8). d , Purified APC/C Cdc20-Hsl1 ternary complex on SDS-PAGE. e , A typical cryo-EM micrograph of APC/C Cdc20-Hsl1 representative of 15,582 micrographs. f , Gallery of two-dimensional averages of APC/C Cdc20-Hsl1 showing different views; representative of 100 two-dimensional averages. g for gel source data.

    Article Snippet: Harvested cell pellets were resuspended in Cdc20 lysis buffer (50 mM Hepes pH 7.8, 500 mM NaCl, 30 mM imidazole, 10% glycerol and 0.5 mM TCEP) supplemented with 0.1 mM PMSF, 5 units/ml benzonase and CompleteTM EDTA-free protease inhibitors and loaded onto a HisTrap HP column (GE Healthcare).

    Techniques: Recombinant, In Vitro, SDS Page, Activation Assay, Purification, Mutagenesis

    Comparison of apo APC/C in unphosphorylated and phosphorylated states. a , b , Superposition of the apo unphosphorylated (magenta) and phosphorylated (cyan) APC/C EM maps revealed little conformational differences except in the vicinity of the C-box binding site. c , Apc3A is in an equilibrium between open (light blue) and closed (orange) conformations. While in the inactive apo state, the majority of Apc3A is in the closed state, association of Cdc20 IR stabilizes the open state. d , Sequence alignment of the Apc1 300s loop across different species human, mouse, Xenopus tropicalis (Western clawed frog) and Danio rerio (zebrafish). Phosphorylation sites are indicated and residues 361-380 accounting for the Apc1 AI segment are boxed.

    Journal: Nature

    Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

    doi: 10.1038/nature17973

    Figure Lengend Snippet: Comparison of apo APC/C in unphosphorylated and phosphorylated states. a , b , Superposition of the apo unphosphorylated (magenta) and phosphorylated (cyan) APC/C EM maps revealed little conformational differences except in the vicinity of the C-box binding site. c , Apc3A is in an equilibrium between open (light blue) and closed (orange) conformations. While in the inactive apo state, the majority of Apc3A is in the closed state, association of Cdc20 IR stabilizes the open state. d , Sequence alignment of the Apc1 300s loop across different species human, mouse, Xenopus tropicalis (Western clawed frog) and Danio rerio (zebrafish). Phosphorylation sites are indicated and residues 361-380 accounting for the Apc1 AI segment are boxed.

    Article Snippet: Harvested cell pellets were resuspended in Cdc20 lysis buffer (50 mM Hepes pH 7.8, 500 mM NaCl, 30 mM imidazole, 10% glycerol and 0.5 mM TCEP) supplemented with 0.1 mM PMSF, 5 units/ml benzonase and CompleteTM EDTA-free protease inhibitors and loaded onto a HisTrap HP column (GE Healthcare).

    Techniques: Binding Assay, Sequencing, Western Blot

    Analytical gel filtration and activity assays. a , With equal amount of input Cdc20, phosphorylated APC/C could form a stable binary complex with Cdc20 after a gel filtration purification step (lane 5), whereas unphosphorylated APC/C could not (lane 4). b , Both unphosphorylated and phosphorylated APC/C associate with Cdh1 stably on gel filtration, as well as APC/C ΔApc1-300s ) served as a loading control and unphosphorylated APC/C alone is used as a negative control for Western-blotting. c , Point mutations of peptide 7 (residues 361-380), either when Arg368 was mutated to glutamate or when the four neighbouring serines were mutated to phospho-mimics (Glu), caused the peptide to abolish its inhibition effect and restored the APC/C activity (lanes 4, 5). Phosphorylation of a single Ser377 only resulted in partial activation of the APC/C (lane 6). d , Chimeric proteins composed of the NTD, the WD40 domain and the IR tail of either Cdc20 or Cdh1 were purified to study their differences in APC/C activation. Both the NTD and the CTD of the coactivators are essential for their association with the APC/C. Swapping both NTD and CTD of Cdh1 with Cdc20 makes it phosphorylation sensitive (lanes 7, 8), similar to Cdc20 (lanes 9, 10) and vice versa . e , Upper panel: Cdh1 can activate both unphosphorylated and phosphorylated APC/C similarly, whereas Cdc20 requires APC/C phosphorylation for its activity. Lower panel: A titration of Cdh1 against unphosphorylated APC/C and APC/C ΔApc1-300s showed enhanced activity in the absence of the Apc1 AI segment at low Cdh1 concentration (≤ 10 nM), whereas Cdc20 requires displacement of the AI segment for its activity. f , Deletion of the Cdh1 α3-helix resulted in reduced activation of the APC/C and makes Cdh1 more phosphorylation sensitive. The substrate Cdk2-cyclin A2-Cks2 was used for assay in c and Hsl1 for the assays in d-f . 20 nM Cdc20 was used in c , 10 nM chimeric coactivators in d and 30 nM coactivators in f . Experiments in a and b were replicated two times, in c, e and f three times and in d for gel source data.

    Journal: Nature

    Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

    doi: 10.1038/nature17973

    Figure Lengend Snippet: Analytical gel filtration and activity assays. a , With equal amount of input Cdc20, phosphorylated APC/C could form a stable binary complex with Cdc20 after a gel filtration purification step (lane 5), whereas unphosphorylated APC/C could not (lane 4). b , Both unphosphorylated and phosphorylated APC/C associate with Cdh1 stably on gel filtration, as well as APC/C ΔApc1-300s ) served as a loading control and unphosphorylated APC/C alone is used as a negative control for Western-blotting. c , Point mutations of peptide 7 (residues 361-380), either when Arg368 was mutated to glutamate or when the four neighbouring serines were mutated to phospho-mimics (Glu), caused the peptide to abolish its inhibition effect and restored the APC/C activity (lanes 4, 5). Phosphorylation of a single Ser377 only resulted in partial activation of the APC/C (lane 6). d , Chimeric proteins composed of the NTD, the WD40 domain and the IR tail of either Cdc20 or Cdh1 were purified to study their differences in APC/C activation. Both the NTD and the CTD of the coactivators are essential for their association with the APC/C. Swapping both NTD and CTD of Cdh1 with Cdc20 makes it phosphorylation sensitive (lanes 7, 8), similar to Cdc20 (lanes 9, 10) and vice versa . e , Upper panel: Cdh1 can activate both unphosphorylated and phosphorylated APC/C similarly, whereas Cdc20 requires APC/C phosphorylation for its activity. Lower panel: A titration of Cdh1 against unphosphorylated APC/C and APC/C ΔApc1-300s showed enhanced activity in the absence of the Apc1 AI segment at low Cdh1 concentration (≤ 10 nM), whereas Cdc20 requires displacement of the AI segment for its activity. f , Deletion of the Cdh1 α3-helix resulted in reduced activation of the APC/C and makes Cdh1 more phosphorylation sensitive. The substrate Cdk2-cyclin A2-Cks2 was used for assay in c and Hsl1 for the assays in d-f . 20 nM Cdc20 was used in c , 10 nM chimeric coactivators in d and 30 nM coactivators in f . Experiments in a and b were replicated two times, in c, e and f three times and in d for gel source data.

    Article Snippet: Harvested cell pellets were resuspended in Cdc20 lysis buffer (50 mM Hepes pH 7.8, 500 mM NaCl, 30 mM imidazole, 10% glycerol and 0.5 mM TCEP) supplemented with 0.1 mM PMSF, 5 units/ml benzonase and CompleteTM EDTA-free protease inhibitors and loaded onto a HisTrap HP column (GE Healthcare).

    Techniques: Filtration, Activity Assay, Purification, Stable Transfection, Negative Control, Western Blot, Inhibition, Activation Assay, Titration, Concentration Assay

    EM reconstructions of the APC/C Cdc20-Hsl1 complex and comparison of Cdc20 NTD and Cdh1 NTD . a, b , Two views of APC/C Cdc20-Hsl1 shown in cartoon with the D box and Cdc20 IR highlighted in surface representation. Cdc20 binds to the APC/C in juxtaposition to Apc10 to form the substrate recognition module. Apc11 is modelled based on the APC/C Cdh1-Emi1 . c , Both Cdc20 NTD (purple) and Cdh1 NTD (grey, aligned to APC/C Cdc20-Hsl1 interact with Apc1 and Apc8B , whereas Cdh1 NTD contains an additional α3-helix associating with Apc1. I) The crucial C box motif is well conserved between the two coactivators and forms extensive interactions with Apc8B. II) The KLLR motif of Cdh1 is present in the α3-helix to engage Apc1, whereas the related Cdc20 KILR motif contacts Apc8B to augment C-box binding.

    Journal: Nature

    Article Title: Molecular mechanism of APC/C activation by mitotic phosphorylation

    doi: 10.1038/nature17973

    Figure Lengend Snippet: EM reconstructions of the APC/C Cdc20-Hsl1 complex and comparison of Cdc20 NTD and Cdh1 NTD . a, b , Two views of APC/C Cdc20-Hsl1 shown in cartoon with the D box and Cdc20 IR highlighted in surface representation. Cdc20 binds to the APC/C in juxtaposition to Apc10 to form the substrate recognition module. Apc11 is modelled based on the APC/C Cdh1-Emi1 . c , Both Cdc20 NTD (purple) and Cdh1 NTD (grey, aligned to APC/C Cdc20-Hsl1 interact with Apc1 and Apc8B , whereas Cdh1 NTD contains an additional α3-helix associating with Apc1. I) The crucial C box motif is well conserved between the two coactivators and forms extensive interactions with Apc8B. II) The KLLR motif of Cdh1 is present in the α3-helix to engage Apc1, whereas the related Cdc20 KILR motif contacts Apc8B to augment C-box binding.

    Article Snippet: Harvested cell pellets were resuspended in Cdc20 lysis buffer (50 mM Hepes pH 7.8, 500 mM NaCl, 30 mM imidazole, 10% glycerol and 0.5 mM TCEP) supplemented with 0.1 mM PMSF, 5 units/ml benzonase and CompleteTM EDTA-free protease inhibitors and loaded onto a HisTrap HP column (GE Healthcare).

    Techniques: Cytotoxicity Assay, Binding Assay