Structured Review

GE Healthcare gst fusion proteins
CD63 participates in HCV entry through a direct interaction with HCV E2. (A) Co-immunoprecipitations of CD63 and CD81 by HCV E2. Extracts of Huh 7.5.1 cells, which were infected with JC1 E2 FLAG virus (MOI 0.3) for 72 hrs, were analyzed by Western blotting to detect the indicated proteins before and after immunoprecipitations with a FLAG antibody or a control mouse antibody. Actin is a negative control. (B) <t>GST</t> pull-down assays with purified proteins. GST-fused CD63 EC2 (GST-CD63) and GST-fused CD81 LEL (GST-CD81) proteins were expressed in E. coli and then purified ( Methods ). FLAG-tagged HCV E2 (FLAG-E2) proteins were expressed in yeast and then purified ( Methods ). After incubating the purified FLAG-E2 proteins with GST, GST-CD81 or GST-CD63 proteins for 2 hrs at 4°C, GST, GST-fusion proteins, and their associated proteins were precipitated with GSH <t>Sepharose</t> 4B. The resin-bound proteins were analyzed by Western blotting with antibodies against GST or FLAG. Degraded forms of GST-CD63 and GST-CD81 (indicated by asterisks) were also precipitated by the GSH resin. (C) Effect of a polypeptide corresponding to the CD63 EC2 domain on HCV infection. JFH1 5A-Rluc virus was incubated with GST or GST-CD63 for 2 hrs at 4°C. Huh7.5.1 cells were then inoculated with the virus–polypeptide mixtures by incubating for 3 hrs at 37°C, and the cells were further cultivated for 48 hrs. Virus infectivity was monitored by measuring Renilla luciferase activities in cell extracts, and normalized to the amounts of proteins in cell extracts (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted. (D) Effect of an anti-CD63 antibody (BEM-1 from Santa Cruz Biotechnologies) on HCV infection. Huh7.5.1 cells were pre-incubated with a negative control mouse IgG1, a positive control anti-CD81 antibody, or an anti-CD63 antibody at the indicated concentrations for 1 hour at 37°C, and then inoculated with JFH1 5A-Rluc virus (MOI of 0.3). The cells were cultivated for additional 48 hrs, and then Renilla luciferase activities in cell lysates were measured and normalized to the amounts of proteins in lysates (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted.
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1) Product Images from "Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics"

Article Title: Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060333

CD63 participates in HCV entry through a direct interaction with HCV E2. (A) Co-immunoprecipitations of CD63 and CD81 by HCV E2. Extracts of Huh 7.5.1 cells, which were infected with JC1 E2 FLAG virus (MOI 0.3) for 72 hrs, were analyzed by Western blotting to detect the indicated proteins before and after immunoprecipitations with a FLAG antibody or a control mouse antibody. Actin is a negative control. (B) GST pull-down assays with purified proteins. GST-fused CD63 EC2 (GST-CD63) and GST-fused CD81 LEL (GST-CD81) proteins were expressed in E. coli and then purified ( Methods ). FLAG-tagged HCV E2 (FLAG-E2) proteins were expressed in yeast and then purified ( Methods ). After incubating the purified FLAG-E2 proteins with GST, GST-CD81 or GST-CD63 proteins for 2 hrs at 4°C, GST, GST-fusion proteins, and their associated proteins were precipitated with GSH Sepharose 4B. The resin-bound proteins were analyzed by Western blotting with antibodies against GST or FLAG. Degraded forms of GST-CD63 and GST-CD81 (indicated by asterisks) were also precipitated by the GSH resin. (C) Effect of a polypeptide corresponding to the CD63 EC2 domain on HCV infection. JFH1 5A-Rluc virus was incubated with GST or GST-CD63 for 2 hrs at 4°C. Huh7.5.1 cells were then inoculated with the virus–polypeptide mixtures by incubating for 3 hrs at 37°C, and the cells were further cultivated for 48 hrs. Virus infectivity was monitored by measuring Renilla luciferase activities in cell extracts, and normalized to the amounts of proteins in cell extracts (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted. (D) Effect of an anti-CD63 antibody (BEM-1 from Santa Cruz Biotechnologies) on HCV infection. Huh7.5.1 cells were pre-incubated with a negative control mouse IgG1, a positive control anti-CD81 antibody, or an anti-CD63 antibody at the indicated concentrations for 1 hour at 37°C, and then inoculated with JFH1 5A-Rluc virus (MOI of 0.3). The cells were cultivated for additional 48 hrs, and then Renilla luciferase activities in cell lysates were measured and normalized to the amounts of proteins in lysates (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted.
Figure Legend Snippet: CD63 participates in HCV entry through a direct interaction with HCV E2. (A) Co-immunoprecipitations of CD63 and CD81 by HCV E2. Extracts of Huh 7.5.1 cells, which were infected with JC1 E2 FLAG virus (MOI 0.3) for 72 hrs, were analyzed by Western blotting to detect the indicated proteins before and after immunoprecipitations with a FLAG antibody or a control mouse antibody. Actin is a negative control. (B) GST pull-down assays with purified proteins. GST-fused CD63 EC2 (GST-CD63) and GST-fused CD81 LEL (GST-CD81) proteins were expressed in E. coli and then purified ( Methods ). FLAG-tagged HCV E2 (FLAG-E2) proteins were expressed in yeast and then purified ( Methods ). After incubating the purified FLAG-E2 proteins with GST, GST-CD81 or GST-CD63 proteins for 2 hrs at 4°C, GST, GST-fusion proteins, and their associated proteins were precipitated with GSH Sepharose 4B. The resin-bound proteins were analyzed by Western blotting with antibodies against GST or FLAG. Degraded forms of GST-CD63 and GST-CD81 (indicated by asterisks) were also precipitated by the GSH resin. (C) Effect of a polypeptide corresponding to the CD63 EC2 domain on HCV infection. JFH1 5A-Rluc virus was incubated with GST or GST-CD63 for 2 hrs at 4°C. Huh7.5.1 cells were then inoculated with the virus–polypeptide mixtures by incubating for 3 hrs at 37°C, and the cells were further cultivated for 48 hrs. Virus infectivity was monitored by measuring Renilla luciferase activities in cell extracts, and normalized to the amounts of proteins in cell extracts (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted. (D) Effect of an anti-CD63 antibody (BEM-1 from Santa Cruz Biotechnologies) on HCV infection. Huh7.5.1 cells were pre-incubated with a negative control mouse IgG1, a positive control anti-CD81 antibody, or an anti-CD63 antibody at the indicated concentrations for 1 hour at 37°C, and then inoculated with JFH1 5A-Rluc virus (MOI of 0.3). The cells were cultivated for additional 48 hrs, and then Renilla luciferase activities in cell lysates were measured and normalized to the amounts of proteins in lysates (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted.

Techniques Used: Infection, Western Blot, Negative Control, Purification, Incubation, Luciferase, Positive Control

2) Product Images from "The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation"

Article Title: The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1002480

The structure of the TPR domain of Rrp5. (A) Cartoon representation of the crystal structure. (B) Surface charge distribution of the TPR domain of Rrp5. (C) Surface representation of amino acid conservation calculated using the ConSurf server [ 35 ] and displayed in PyMOL (The PyMOL Molecular Graphics System, Version 1.0r1 Schrödinger). The program ConSurf uses the provided sequence ( S . cerevisiae Rrp5) to find the 150 closest homologs (lowest E-values) for generating a multiple sequence alignment. The organisms used for Rrp5-TPR can be found here: http://consurf.tau.ac.il/results/1463756501/query_final_homolougs.html . The conserved patches on the surface of the TPR motif that interact with Rok1 are marked in red. (D) Mutations in conserved charged areas of the TPR domain are deleterious in vivo. TPR2: E1509K/E1510K/E1512K and TPR7: K1686E/K1689E. (E) Mutations in conserved charged areas of the TPR domain weaken the Rok1•Rrp5 interaction in vitro. (F) Position of the TPR mutations in the 3-D reconstruction of the Rrp5•Rok1 complex. (G) Recombinant GST-Rok1 immobilized on glutathione (GSH) sepharose resin interacts with recombinant, purified Rrp5 FL , Rrp5_C7, and Rrp5_C6, but not Rrp5_C5. (H) Maltose binding protein (MBP) or glutathione S-transferase (GST) alone does not bind to the Rrp5 fragments tested here. I, input; W, wash; E, elution.
Figure Legend Snippet: The structure of the TPR domain of Rrp5. (A) Cartoon representation of the crystal structure. (B) Surface charge distribution of the TPR domain of Rrp5. (C) Surface representation of amino acid conservation calculated using the ConSurf server [ 35 ] and displayed in PyMOL (The PyMOL Molecular Graphics System, Version 1.0r1 Schrödinger). The program ConSurf uses the provided sequence ( S . cerevisiae Rrp5) to find the 150 closest homologs (lowest E-values) for generating a multiple sequence alignment. The organisms used for Rrp5-TPR can be found here: http://consurf.tau.ac.il/results/1463756501/query_final_homolougs.html . The conserved patches on the surface of the TPR motif that interact with Rok1 are marked in red. (D) Mutations in conserved charged areas of the TPR domain are deleterious in vivo. TPR2: E1509K/E1510K/E1512K and TPR7: K1686E/K1689E. (E) Mutations in conserved charged areas of the TPR domain weaken the Rok1•Rrp5 interaction in vitro. (F) Position of the TPR mutations in the 3-D reconstruction of the Rrp5•Rok1 complex. (G) Recombinant GST-Rok1 immobilized on glutathione (GSH) sepharose resin interacts with recombinant, purified Rrp5 FL , Rrp5_C7, and Rrp5_C6, but not Rrp5_C5. (H) Maltose binding protein (MBP) or glutathione S-transferase (GST) alone does not bind to the Rrp5 fragments tested here. I, input; W, wash; E, elution.

Techniques Used: Sequencing, In Vivo, In Vitro, Recombinant, Purification, Binding Assay

The structure of Rrp5 complexes. 3-D reconstructions of (A) Rrp5•Rok1 or (B) Rrp5•RNA. 90° rotations of Rrp5 complexes around its long axis show the curved, thumb-like projection and hollow fist-like body. The area corresponding to Rok1’s RecA motifs is shaded in blue. By Fourier shell correlation (FSC) = 0.5, the structures are each at about 2.8 nm resolution ( S9A Fig ). In the final column, the asterisk marks the tip of the TPR.
Figure Legend Snippet: The structure of Rrp5 complexes. 3-D reconstructions of (A) Rrp5•Rok1 or (B) Rrp5•RNA. 90° rotations of Rrp5 complexes around its long axis show the curved, thumb-like projection and hollow fist-like body. The area corresponding to Rok1’s RecA motifs is shaded in blue. By Fourier shell correlation (FSC) = 0.5, the structures are each at about 2.8 nm resolution ( S9A Fig ). In the final column, the asterisk marks the tip of the TPR.

Techniques Used:

Rrp5 binds to 40S ribosomal subunits in vivo and in vitro. (A) Ultracentrifugation pelleting experiments demonstrate that recombinant full-length Rrp5 binds purified mature 40S subunits in vitro. S, supernatant; P, pellet. (B) The three C-terminal S1 domains are essential for the interaction between Rrp5 and 40S subunits in vitro. Recombinant Rrp5 fragments are used in the same pelleting experiment as 1A. (C) Gradient centrifugation demonstrates a role for the three C-terminal S1 domains and S1–S5 for binding to preribosomes in vivo. (D) Gradient sedimentation experiments of various recombinant Rrp5•Rok1•AMPPCP complexes demonstrate that S1–S5 contributes to Rrp5 binding to 40S ribosomes in vitro. Note that the more sensitive gradient sedimentation experiments are required to demonstrate the more quantitative than qualitative differences in Rrp8_C8 and Rrp5_C7 binding. Because Rrp5_N9 and Rrp5_N12 do not bind Rok1, binding of these fragments requires the pelleting assay. All experiments were repeated at least twice, and representative data are shown.
Figure Legend Snippet: Rrp5 binds to 40S ribosomal subunits in vivo and in vitro. (A) Ultracentrifugation pelleting experiments demonstrate that recombinant full-length Rrp5 binds purified mature 40S subunits in vitro. S, supernatant; P, pellet. (B) The three C-terminal S1 domains are essential for the interaction between Rrp5 and 40S subunits in vitro. Recombinant Rrp5 fragments are used in the same pelleting experiment as 1A. (C) Gradient centrifugation demonstrates a role for the three C-terminal S1 domains and S1–S5 for binding to preribosomes in vivo. (D) Gradient sedimentation experiments of various recombinant Rrp5•Rok1•AMPPCP complexes demonstrate that S1–S5 contributes to Rrp5 binding to 40S ribosomes in vitro. Note that the more sensitive gradient sedimentation experiments are required to demonstrate the more quantitative than qualitative differences in Rrp8_C8 and Rrp5_C7 binding. Because Rrp5_N9 and Rrp5_N12 do not bind Rok1, binding of these fragments requires the pelleting assay. All experiments were repeated at least twice, and representative data are shown.

Techniques Used: In Vivo, In Vitro, Recombinant, Purification, Gradient Centrifugation, Binding Assay, Sedimentation

ATP-hydrolysis by Rok1 is required for Rrp5 release from pre-40S ribosomes in vivo. (A) Western and northern blot analysis of pre-40S ribosomes purified from yeast cells with Enp1-TAP reveals the accumulation of Rrp5, Has1, and snR30 in pre-40S complexes isolated from Rok1 ATPase-deficient cells relative to the WT cells. The TAP-antibody was used as a loading control. (B) Northern blot analysis of preribosomes captured with Rrp5-TAP in the presence of wild-type Rok1 (WT) or ATPase-deficient mutants of Rok1 (K172A and D280A) reveals decreased binding to pre-60S subunits (containing 27S pre-rRNAs) relative to pre-40S subunits (containing 23S pre-rRNA). The data from three biological replicates were quantitated and normalized such that in wild-type cells the 27S/23S levels are set to 1 and the mutants are expressed relative to the WT. The numerical data underlying this graph can be found in S1 Data .
Figure Legend Snippet: ATP-hydrolysis by Rok1 is required for Rrp5 release from pre-40S ribosomes in vivo. (A) Western and northern blot analysis of pre-40S ribosomes purified from yeast cells with Enp1-TAP reveals the accumulation of Rrp5, Has1, and snR30 in pre-40S complexes isolated from Rok1 ATPase-deficient cells relative to the WT cells. The TAP-antibody was used as a loading control. (B) Northern blot analysis of preribosomes captured with Rrp5-TAP in the presence of wild-type Rok1 (WT) or ATPase-deficient mutants of Rok1 (K172A and D280A) reveals decreased binding to pre-60S subunits (containing 27S pre-rRNAs) relative to pre-40S subunits (containing 23S pre-rRNA). The data from three biological replicates were quantitated and normalized such that in wild-type cells the 27S/23S levels are set to 1 and the mutants are expressed relative to the WT. The numerical data underlying this graph can be found in S1 Data .

Techniques Used: In Vivo, Western Blot, Northern Blot, Purification, Isolation, Binding Assay

Has1 and snR30 accumulate in pre-40S subunits when the Rok1•Rrp5 interaction is disrupted. (A) Western and northern blot analysis of pre-40S ribosomes purified with Enp1-TAP from yeast cells expressing wild-type Rrp5 or TPR2+7-mutant Rrp5. The TAP-antibody was used as a loading control. (B) Quantitation of two biological replicates of data on the left. The difference in copurification of the named AFs with mutant and wild-type Rrp5 is shown. The numerical data underlying this graph can be found in S1 Data . (C) Recombinant Has1 binds recombinant His 6 -Rrp5, but not recombinant MBP-Rok1, and recombinant MBP-Has1 does not bind recombinant Rok1. Has1 alone does not bind to the Ni beads.
Figure Legend Snippet: Has1 and snR30 accumulate in pre-40S subunits when the Rok1•Rrp5 interaction is disrupted. (A) Western and northern blot analysis of pre-40S ribosomes purified with Enp1-TAP from yeast cells expressing wild-type Rrp5 or TPR2+7-mutant Rrp5. The TAP-antibody was used as a loading control. (B) Quantitation of two biological replicates of data on the left. The difference in copurification of the named AFs with mutant and wild-type Rrp5 is shown. The numerical data underlying this graph can be found in S1 Data . (C) Recombinant Has1 binds recombinant His 6 -Rrp5, but not recombinant MBP-Rok1, and recombinant MBP-Has1 does not bind recombinant Rok1. Has1 alone does not bind to the Ni beads.

Techniques Used: Western Blot, Northern Blot, Purification, Expressing, Mutagenesis, Quantitation Assay, Copurification, Recombinant

Speculative model for Rrp5•Rok1 function during 40S ribosome maturation. Rrp5 (in magenta) is bound to pre-40S subunits (green) and interacts with ITS1 (grey). Rok1 (in yellow) is recruited to pre-40S subunits (I),and then forms the inhibitory duplex to block premature 3′-end formation (II, different secondary structure shown in lighter grey, indicated by asterisk). After cleavage in ITS1 (III, marked by arrow), ATP (T) is hydrolyzed to ADP (D, IV), leading to dissociation of Rrp5•Rok1 from pre-40S subunits (V).
Figure Legend Snippet: Speculative model for Rrp5•Rok1 function during 40S ribosome maturation. Rrp5 (in magenta) is bound to pre-40S subunits (green) and interacts with ITS1 (grey). Rok1 (in yellow) is recruited to pre-40S subunits (I),and then forms the inhibitory duplex to block premature 3′-end formation (II, different secondary structure shown in lighter grey, indicated by asterisk). After cleavage in ITS1 (III, marked by arrow), ATP (T) is hydrolyzed to ADP (D, IV), leading to dissociation of Rrp5•Rok1 from pre-40S subunits (V).

Techniques Used: Blocking Assay

Rok1•Rrp5 binds to 40S subunits in the ATP-bound form. (A) Glycerol density gradient followed by protein precipitation and western blotting was used to analyze the binding of recombinant Rrp5•Rok1 to 40S subunits in the presence of AMPPCP or ADP. Fractions 1 and 13 represent the top (5% glycerol) and the bottom (20% glycerol) of the gradient, respectively. (B) Quantitation of the data in panel A and S2B Fig . The data are averages from two biological replicates. The numerical data underlying this graph can be found in S1 Data . (C) In the absence of 40S ribosomes, neither Rok1 nor Rrp5 enter the gradient, demonstrating that cosedimentation with 40S ribosomes reflects 40S binding. (D) Control gradients in the absence of Rok1 demonstrate that the AMPPCP- and ADP-dependent effects are mediated by Rok1. (E) Rok1 alone does not bind to 40S subunits in the presence of either AMPPCP or ADP. All experiments were repeated at least twice, and representative gels are shown. (F) Electrophoretic mobility shift assay of Rrp5_C7•Rok1 to H44-A 2 rRNA mimics in the presence of AMPPCP or ADP. The left panel shows quantitation of data on the right, fit with a single binding isotherm to yield K 1/2 values for the AMPPCP and ADP states of 57 ± 7 nM and 106 ± 24 nM, respectively. The numerical data underlying this graph can be found in S1 Data . Error bars come from three independent experiments.
Figure Legend Snippet: Rok1•Rrp5 binds to 40S subunits in the ATP-bound form. (A) Glycerol density gradient followed by protein precipitation and western blotting was used to analyze the binding of recombinant Rrp5•Rok1 to 40S subunits in the presence of AMPPCP or ADP. Fractions 1 and 13 represent the top (5% glycerol) and the bottom (20% glycerol) of the gradient, respectively. (B) Quantitation of the data in panel A and S2B Fig . The data are averages from two biological replicates. The numerical data underlying this graph can be found in S1 Data . (C) In the absence of 40S ribosomes, neither Rok1 nor Rrp5 enter the gradient, demonstrating that cosedimentation with 40S ribosomes reflects 40S binding. (D) Control gradients in the absence of Rok1 demonstrate that the AMPPCP- and ADP-dependent effects are mediated by Rok1. (E) Rok1 alone does not bind to 40S subunits in the presence of either AMPPCP or ADP. All experiments were repeated at least twice, and representative gels are shown. (F) Electrophoretic mobility shift assay of Rrp5_C7•Rok1 to H44-A 2 rRNA mimics in the presence of AMPPCP or ADP. The left panel shows quantitation of data on the right, fit with a single binding isotherm to yield K 1/2 values for the AMPPCP and ADP states of 57 ± 7 nM and 106 ± 24 nM, respectively. The numerical data underlying this graph can be found in S1 Data . Error bars come from three independent experiments.

Techniques Used: Western Blot, Binding Assay, Recombinant, Quantitation Assay, Electrophoretic Mobility Shift Assay

3) Product Images from "Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway"

Article Title: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1000763

Sae2 protein interacts with PBD of Cdc5. (A) Sae2 protein sequence. The putative Cdc5 phosphorylation sites and PBD binding sites are indicated. (B) Plasmid pEG202-PBD 340–705 , carrying the polo box domain of Cdc5 (PBD, aa 340 to 705), and pJG4-5-SAE2, carrying the full length SAE2 gene under the GAL1 promoter, were co-transformed with pSH18-34, a β-galactosidase reporter plasmid in the wild type yeast strain EGY48. To assess two-hybrid interaction, these strains were patched on to 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X Gal) plates containing either raffinose (RAF, prey repressed) or galactose (GAL, prey expressed). Accordingly to [50] , the strain Y692 (PBD versus Swe1 173–400 protein fragment) was used as positive control. (C) Cells of the strain Y202, expressing SAE2 -3HA gene, were blocked in G2/M by nocodazole treatment. Whole cell protein extract was prepared and incubated with glutathione-Sepharose beads carrying GST or GST-PBD 357–705 . Input and pull-down samples were analyzed by western blotting with monoclonal antibody 12CA5 (αHA) or polyclonal antisera raised against GST (αGST). Asterisk denotes bands of GST-PBD degradation or expression of truncated proteins. (D) Schematic model to summarize the results presented in this work. (i) Sae2 transiently binds DSB, regulating ends resection and influencing Mec1-signaling. The checkpoint signal is amplified downstream, regulating several targets, including Cdc5. (ii) After a prolonged checkpoint response, adaptation to damage takes over and Cdc5 is re-activated, likely by an activating kinase (in human cells, it is aurora A [35] ); Cdc5 then inhibits checkpoint signaling in a feedback regulatory loop, by likely targeting several factors, including Sae2 whose loading on the irreparable DSB increases, slowing down resection and contributing to counteract the checkpoint signaling (red circles denote phosphorylation). Alternatively, or in addition, Cdc5 function on several targets, including Sae2, is enhanced in the presence of elevated levels of Cdc5, a situation frequently found for Plks in tumor cells.
Figure Legend Snippet: Sae2 protein interacts with PBD of Cdc5. (A) Sae2 protein sequence. The putative Cdc5 phosphorylation sites and PBD binding sites are indicated. (B) Plasmid pEG202-PBD 340–705 , carrying the polo box domain of Cdc5 (PBD, aa 340 to 705), and pJG4-5-SAE2, carrying the full length SAE2 gene under the GAL1 promoter, were co-transformed with pSH18-34, a β-galactosidase reporter plasmid in the wild type yeast strain EGY48. To assess two-hybrid interaction, these strains were patched on to 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X Gal) plates containing either raffinose (RAF, prey repressed) or galactose (GAL, prey expressed). Accordingly to [50] , the strain Y692 (PBD versus Swe1 173–400 protein fragment) was used as positive control. (C) Cells of the strain Y202, expressing SAE2 -3HA gene, were blocked in G2/M by nocodazole treatment. Whole cell protein extract was prepared and incubated with glutathione-Sepharose beads carrying GST or GST-PBD 357–705 . Input and pull-down samples were analyzed by western blotting with monoclonal antibody 12CA5 (αHA) or polyclonal antisera raised against GST (αGST). Asterisk denotes bands of GST-PBD degradation or expression of truncated proteins. (D) Schematic model to summarize the results presented in this work. (i) Sae2 transiently binds DSB, regulating ends resection and influencing Mec1-signaling. The checkpoint signal is amplified downstream, regulating several targets, including Cdc5. (ii) After a prolonged checkpoint response, adaptation to damage takes over and Cdc5 is re-activated, likely by an activating kinase (in human cells, it is aurora A [35] ); Cdc5 then inhibits checkpoint signaling in a feedback regulatory loop, by likely targeting several factors, including Sae2 whose loading on the irreparable DSB increases, slowing down resection and contributing to counteract the checkpoint signaling (red circles denote phosphorylation). Alternatively, or in addition, Cdc5 function on several targets, including Sae2, is enhanced in the presence of elevated levels of Cdc5, a situation frequently found for Plks in tumor cells.

Techniques Used: Sequencing, Binding Assay, Plasmid Preparation, Transformation Assay, Positive Control, Expressing, Incubation, Western Blot, Amplification

4) Product Images from "Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane"

Article Title: Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane

Journal: eLife

doi: 10.7554/eLife.19394

Binding of purified SNAP25 constructs to reconstituted liposomes. Atto647N-labeled liposomes containing either POPC or a complex lipid mixture (POPC/DOPS/POPE/cholesterol/PI) and the indicated amounts of PI(4,5)P 2 or PI(3,4,5)P 3 were added to immobilized GST-wtSNAP25 (+ 3 ), GST-SNAP25 -5 and GST-SNAP25+ 10 and incubated 1 hr at 4°C. After washing the beads, the amount of bound liposomes was measured by their Atto647N fluorescence (excitation: 639 nm, emission: 669 nm). The amounts of liposomes specifically bound to the different GST-SNAP25 constructs were determined by subtracting the values derived from the GST controls. Values are given as means ± S.E.M. (n = 3; t-test *p
Figure Legend Snippet: Binding of purified SNAP25 constructs to reconstituted liposomes. Atto647N-labeled liposomes containing either POPC or a complex lipid mixture (POPC/DOPS/POPE/cholesterol/PI) and the indicated amounts of PI(4,5)P 2 or PI(3,4,5)P 3 were added to immobilized GST-wtSNAP25 (+ 3 ), GST-SNAP25 -5 and GST-SNAP25+ 10 and incubated 1 hr at 4°C. After washing the beads, the amount of bound liposomes was measured by their Atto647N fluorescence (excitation: 639 nm, emission: 669 nm). The amounts of liposomes specifically bound to the different GST-SNAP25 constructs were determined by subtracting the values derived from the GST controls. Values are given as means ± S.E.M. (n = 3; t-test *p

Techniques Used: Binding Assay, Purification, Construct, Labeling, Incubation, Fluorescence, Derivative Assay

5) Product Images from "A metalloproteinase karilysin present in the majority of Tannerella forsythia isolates inhibits all pathways of the complement system"

Article Title: A metalloproteinase karilysin present in the majority of Tannerella forsythia isolates inhibits all pathways of the complement system

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

doi: 10.4049/jimmunol.1101240

Karilysin degrades preferentiallyα-chains of C4 and C5 , and generates biologically active C5a. C4 (A) and C5 (B) (0.2 μM each) were incubated with increasing concentrations of Kly48 or 1.92 μM proKly E136A . The molar ratios of complement
Figure Legend Snippet: Karilysin degrades preferentiallyα-chains of C4 and C5 , and generates biologically active C5a. C4 (A) and C5 (B) (0.2 μM each) were incubated with increasing concentrations of Kly48 or 1.92 μM proKly E136A . The molar ratios of complement

Techniques Used: Incubation

Karilysin inhibits the terminal pathway of complement
Figure Legend Snippet: Karilysin inhibits the terminal pathway of complement

Techniques Used:

Karilysin and gingipains act synergistically
Figure Legend Snippet: Karilysin and gingipains act synergistically

Techniques Used: Activated Clotting Time Assay

Karilysin destroys bactericidal and hemolytic activity of human serum
Figure Legend Snippet: Karilysin destroys bactericidal and hemolytic activity of human serum

Techniques Used: Activity Assay

Karilysin inhibits the classical pathway of complement
Figure Legend Snippet: Karilysin inhibits the classical pathway of complement

Techniques Used:

MBL, ficolin-2 and ficolin-3 are cleaved by karilysin
Figure Legend Snippet: MBL, ficolin-2 and ficolin-3 are cleaved by karilysin

Techniques Used:

Karilysin inhibits the lectin pathway of complement
Figure Legend Snippet: Karilysin inhibits the lectin pathway of complement

Techniques Used:

The gene kly encoding karilysin is commonly present in T. forsythia
Figure Legend Snippet: The gene kly encoding karilysin is commonly present in T. forsythia

Techniques Used:

6) Product Images from "c-Jun N-Terminal Kinase Phosphorylation of MARCKSL1 Determines Actin Stability and Migration in Neurons and in Cancer Cells"

Article Title: c-Jun N-Terminal Kinase Phosphorylation of MARCKSL1 Determines Actin Stability and Migration in Neurons and in Cancer Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00713-12

Identification of brain-derived MARCKSL1 as a JNK substrate. (A) LC-ESI-qTOF MS/MS sequencing identified this protein as a MARCKS-related protein (MARCKSL1). Identified peptides are shown. (B) Bacterially expressed GST-MARCKSL1 was phosphorylated by active
Figure Legend Snippet: Identification of brain-derived MARCKSL1 as a JNK substrate. (A) LC-ESI-qTOF MS/MS sequencing identified this protein as a MARCKS-related protein (MARCKSL1). Identified peptides are shown. (B) Bacterially expressed GST-MARCKSL1 was phosphorylated by active

Techniques Used: Derivative Assay, Mass Spectrometry, Sequencing

7) Product Images from "Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase"

Article Title: Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase

Journal: Journal of Clinical Investigation

doi:

Confirmation of AMPK-CFTR interaction by GST pull-down assays. ( a ) CFTR COOH-terminal tail (GST-CFTR-1411–1480) pulls down α1-AMPK in vitro. Purified liver AMPK holoenzyme (15 ng) was loaded in first lane as reference. ( b ) GST-α1-AMPK fusion protein expressed in CHO-BQ2 cells pulls down full-length CFTR in vivo. Ten micrograms of the total soluble cellular protein was loaded in first and third (lysate) lanes as reference for CFTR. Second and fourth (beads) lanes show eluate from GSH-Sepharose beads after affinity purification and washing. ( c ) Comparison of binding strengths of CFTR with NH 2 -terminal and COOH-terminal GST-α1-AMPK fusion proteins in CHO-BQ2 cells. Lower panel is the same membrane probed with anti-GST antibodies to allow comparisons of different CFTR bands in panel above. ( d ) GST-α2-AMPK fusion protein expressed in CHO-BQ2 cells pulls down CFTR in vivo. All results shown are representative of at least three replicate experiments.
Figure Legend Snippet: Confirmation of AMPK-CFTR interaction by GST pull-down assays. ( a ) CFTR COOH-terminal tail (GST-CFTR-1411–1480) pulls down α1-AMPK in vitro. Purified liver AMPK holoenzyme (15 ng) was loaded in first lane as reference. ( b ) GST-α1-AMPK fusion protein expressed in CHO-BQ2 cells pulls down full-length CFTR in vivo. Ten micrograms of the total soluble cellular protein was loaded in first and third (lysate) lanes as reference for CFTR. Second and fourth (beads) lanes show eluate from GSH-Sepharose beads after affinity purification and washing. ( c ) Comparison of binding strengths of CFTR with NH 2 -terminal and COOH-terminal GST-α1-AMPK fusion proteins in CHO-BQ2 cells. Lower panel is the same membrane probed with anti-GST antibodies to allow comparisons of different CFTR bands in panel above. ( d ) GST-α2-AMPK fusion protein expressed in CHO-BQ2 cells pulls down CFTR in vivo. All results shown are representative of at least three replicate experiments.

Techniques Used: In Vitro, Purification, In Vivo, Affinity Purification, Binding Assay

Yeast two-hybrid interaction maps of α1-AMPK and CFTR COOH-terminal tail. ( a ) as template. Other constructs were either original prey clones identified in the two-hybrid screen (α1-161-550 and 294-550) or were generated by PCR using α1-294-550 prey plasmid as a template. ( b ) Determination of region and specific residues within CFTR-1411–1480 important for interaction with α1-294-550. Expression of the CFTR-1420–1443 fragment could not be detected by Western analysis. Because lack of interaction observed may have resulted from no expression, it is uncertain whether residues 1444–1457 are required for strong interaction.
Figure Legend Snippet: Yeast two-hybrid interaction maps of α1-AMPK and CFTR COOH-terminal tail. ( a ) as template. Other constructs were either original prey clones identified in the two-hybrid screen (α1-161-550 and 294-550) or were generated by PCR using α1-294-550 prey plasmid as a template. ( b ) Determination of region and specific residues within CFTR-1411–1480 important for interaction with α1-294-550. Expression of the CFTR-1420–1443 fragment could not be detected by Western analysis. Because lack of interaction observed may have resulted from no expression, it is uncertain whether residues 1444–1457 are required for strong interaction.

Techniques Used: Construct, Clone Assay, Two Hybrid Screening, Generated, Polymerase Chain Reaction, Plasmid Preparation, Expressing, Western Blot

8) Product Images from "Calmodulin in complex with the first IQ motif of myosin-5a functions as an intact calcium sensor"

Article Title: Calmodulin in complex with the first IQ motif of myosin-5a functions as an intact calcium sensor

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

doi: 10.1073/pnas.1607702113

Myo5a-HMM and the MD-IQ1 bind to GST-GTD in a Ca 2+ -dependent manner. FLAG-tagged MD-IQ1 or Myo5a-HMM was incubated with GST-GTD or GST and was pulled down by GSH-Sepharose under EGTA or pCa4 conditions. The pulled-down samples (20 μL each) were
Figure Legend Snippet: Myo5a-HMM and the MD-IQ1 bind to GST-GTD in a Ca 2+ -dependent manner. FLAG-tagged MD-IQ1 or Myo5a-HMM was incubated with GST-GTD or GST and was pulled down by GSH-Sepharose under EGTA or pCa4 conditions. The pulled-down samples (20 μL each) were

Techniques Used: Incubation

CaM continuously associates with the Myo5a MD-IQ1 during the Ca 2+ transition. ( A ) Purified MD-IQ1 bound to anti-Flag beads was washed twice alternately with Ca 2+ -free (EGTA) solution and pCa4 solution, eluted by Flag peptide, and subjected to SDS/PAGE
Figure Legend Snippet: CaM continuously associates with the Myo5a MD-IQ1 during the Ca 2+ transition. ( A ) Purified MD-IQ1 bound to anti-Flag beads was washed twice alternately with Ca 2+ -free (EGTA) solution and pCa4 solution, eluted by Flag peptide, and subjected to SDS/PAGE

Techniques Used: Chick Chorioallantoic Membrane Assay, Purification, SDS Page

9) Product Images from "A transcriptional coactivator, AtGIF1, is involved in regulating leaf growth and morphology in Arabidopsis"

Article Title: A transcriptional coactivator, AtGIF1, is involved in regulating leaf growth and morphology in Arabidopsis

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

doi: 10.1073/pnas.0405450101

In vitro column-binding assay. ( A and B ) HA-tagged full-length GRF1 protein ( A ) or HA-tagged GRF1 fragments ( B ) were applied to a GST or GST-GIF1 column, as indicated. After extensive washing with the binding buffer to remove nonspecifically bound protein, HA-tagged GRF1 or its fragments retained on the column were eluted and subjected to immunoblot analysis with anti-HA antibody. Q, W, and C above the lanes indicate the GRF1 fragments containing the QLQ, WRC, and C-terminal regions, respectively. ( C ) The full-length HA-GRF1 protein was applied to a GST-SNH or GST-QG fusion column followed by immunoblot analysis with anti-HA antibody. “Input” indicates the signal elicited by 1% of applied protein as a control.
Figure Legend Snippet: In vitro column-binding assay. ( A and B ) HA-tagged full-length GRF1 protein ( A ) or HA-tagged GRF1 fragments ( B ) were applied to a GST or GST-GIF1 column, as indicated. After extensive washing with the binding buffer to remove nonspecifically bound protein, HA-tagged GRF1 or its fragments retained on the column were eluted and subjected to immunoblot analysis with anti-HA antibody. Q, W, and C above the lanes indicate the GRF1 fragments containing the QLQ, WRC, and C-terminal regions, respectively. ( C ) The full-length HA-GRF1 protein was applied to a GST-SNH or GST-QG fusion column followed by immunoblot analysis with anti-HA antibody. “Input” indicates the signal elicited by 1% of applied protein as a control.

Techniques Used: In Vitro, Binding Assay

10) Product Images from "Modification of Cry4Aa toward Improved Toxin Processing in the Gut of the Pea Aphid, Acyrthosiphon pisum"

Article Title: Modification of Cry4Aa toward Improved Toxin Processing in the Gut of the Pea Aphid, Acyrthosiphon pisum

Journal: PLoS ONE

doi: 10.1371/journal.pone.0155466

In vitro activation of modified Cry4Aa by aphid gut proteases. Toxins were incubated with the lumen or membrane extracts from the pea aphid gut and hydrolyzed products detected by western blot using Cry4Aa antibodies. Toxin activation profiles were generated in the presence or absence of cathepsin activators (EDTA and cysteine, 3 mM) as indicated. Native Cry4Aa with or without digestion with 5% (w/w) trypsin, and modified Cry4Aa not exposed to proteases or activators were included as controls. Native; native Cry4Aa. Trypsin; native Cry4Aa exposed to 5% w/w trypsin, positive control. Boxes show the faint 20 kDa bands seen on trypsin digestion of native Cry4Aa, and comparison of 45 kDa band lumen fraction degradation products of native Cry4Aa and Cry4Aa 2A toxins.
Figure Legend Snippet: In vitro activation of modified Cry4Aa by aphid gut proteases. Toxins were incubated with the lumen or membrane extracts from the pea aphid gut and hydrolyzed products detected by western blot using Cry4Aa antibodies. Toxin activation profiles were generated in the presence or absence of cathepsin activators (EDTA and cysteine, 3 mM) as indicated. Native Cry4Aa with or without digestion with 5% (w/w) trypsin, and modified Cry4Aa not exposed to proteases or activators were included as controls. Native; native Cry4Aa. Trypsin; native Cry4Aa exposed to 5% w/w trypsin, positive control. Boxes show the faint 20 kDa bands seen on trypsin digestion of native Cry4Aa, and comparison of 45 kDa band lumen fraction degradation products of native Cry4Aa and Cry4Aa 2A toxins.

Techniques Used: In Vitro, Activation Assay, Modification, Incubation, Western Blot, Generated, Positive Control

Impact of Cry4Aa 2A on the aphid midgut epithelium. Light microscope images of guts from aphids fed for 24 h on 10 mM Tris pH 7.5 (control), native Cry4Aa or Cry4Aa 2A in the same buffer. The control image shows a healthy gut epithelium, while epithelia from the toxin treatments show mild (native Cry4Aa) and severe (Cry4a 2A) loss of integrity. Epithelial cells swell, become highly vacuolated, and lyse following toxin binding.
Figure Legend Snippet: Impact of Cry4Aa 2A on the aphid midgut epithelium. Light microscope images of guts from aphids fed for 24 h on 10 mM Tris pH 7.5 (control), native Cry4Aa or Cry4Aa 2A in the same buffer. The control image shows a healthy gut epithelium, while epithelia from the toxin treatments show mild (native Cry4Aa) and severe (Cry4a 2A) loss of integrity. Epithelial cells swell, become highly vacuolated, and lyse following toxin binding.

Techniques Used: Light Microscopy, Binding Assay

Impact of modified Cry4Aa on aphid survival. Pea aphid mortality (%) after one and two days of feeding on Tris buffer, pH 7.5, native Cry4Aa, native trypsin-activated Cry4Aa, and modified Cry4Aa toxins is shown (mean ± SE). Mortality from treatments with different letters on the same day are significantly different (Bonferroni adjustment, p
Figure Legend Snippet: Impact of modified Cry4Aa on aphid survival. Pea aphid mortality (%) after one and two days of feeding on Tris buffer, pH 7.5, native Cry4Aa, native trypsin-activated Cry4Aa, and modified Cry4Aa toxins is shown (mean ± SE). Mortality from treatments with different letters on the same day are significantly different (Bonferroni adjustment, p

Techniques Used: Modification

Engineering of Cry4Aa with cathepsin L and B cleavage sites. Amino acid sequences that are recognized by cathepsin L and B proteases (FR and RR respectively) were added to Cry4Aa or replaced existing amino acids, at two locations. Two additional Cry4Aa constructs were modified at the second region only. See Table 1 for details of the modified toxins produced.
Figure Legend Snippet: Engineering of Cry4Aa with cathepsin L and B cleavage sites. Amino acid sequences that are recognized by cathepsin L and B proteases (FR and RR respectively) were added to Cry4Aa or replaced existing amino acids, at two locations. Two additional Cry4Aa constructs were modified at the second region only. See Table 1 for details of the modified toxins produced.

Techniques Used: Construct, Modification, Produced

Detection of modified Cry4Aa following exposure to cathepsins in the aphid gut. Aphids were fed overnight, and lumen and membrane fractions from dissected guts were separated by SDS-PAGE and analyzed by western blot for toxin profiles. A second replicate of 57 aphid guts yielded similar results in the lumen fraction. Native Cry4Aa controls: diet—Cry4Aa exposed to aphid diet overnight. Sol, Cry4Aa solubilized in PBS pH 7.4. Tryp, native Cry4Aa exposed to 5% w/w trypsin, (positive control). Nat, native Cry4Aa. Note: none of the controls were exposed to aphid gut proteases. Boxes highlight faint 45 and 20 kDa toxin bands.
Figure Legend Snippet: Detection of modified Cry4Aa following exposure to cathepsins in the aphid gut. Aphids were fed overnight, and lumen and membrane fractions from dissected guts were separated by SDS-PAGE and analyzed by western blot for toxin profiles. A second replicate of 57 aphid guts yielded similar results in the lumen fraction. Native Cry4Aa controls: diet—Cry4Aa exposed to aphid diet overnight. Sol, Cry4Aa solubilized in PBS pH 7.4. Tryp, native Cry4Aa exposed to 5% w/w trypsin, (positive control). Nat, native Cry4Aa. Note: none of the controls were exposed to aphid gut proteases. Boxes highlight faint 45 and 20 kDa toxin bands.

Techniques Used: Modification, SDS Page, Western Blot, Positive Control

Trypsin activation of native and modified Cry4A toxins. A. Digestion of native Cry4Aa S1 with 20% trypsin for the specified periods of time resulted in bands of 60, 45 and 20 kDa. The composition of these bands is shown in the schematic diagram at right. Only on prolonged digestion with 20% w/w trypsin (18 and 24 hours) did the 60 kDa intermediate band diminish in intensity. The inset shows the presence of two protein bands in the undigested, purified Cry4Aa-S1, which are assumed to be the 65 and 60 kDa proteins resulting from loss of the N-terminal 36 aa. B. Comparison of the trypsin digestion of the native and modified toxins showed variation in the relative band intensities, with significantly less 60 kDa intermediate in the Cry4Aa 1A and 2A digests. Digestion products separated by SDS-PAGE were transferred to PVDF membrane for western blot detection with anti-Cry4Aa IgG.
Figure Legend Snippet: Trypsin activation of native and modified Cry4A toxins. A. Digestion of native Cry4Aa S1 with 20% trypsin for the specified periods of time resulted in bands of 60, 45 and 20 kDa. The composition of these bands is shown in the schematic diagram at right. Only on prolonged digestion with 20% w/w trypsin (18 and 24 hours) did the 60 kDa intermediate band diminish in intensity. The inset shows the presence of two protein bands in the undigested, purified Cry4Aa-S1, which are assumed to be the 65 and 60 kDa proteins resulting from loss of the N-terminal 36 aa. B. Comparison of the trypsin digestion of the native and modified toxins showed variation in the relative band intensities, with significantly less 60 kDa intermediate in the Cry4Aa 1A and 2A digests. Digestion products separated by SDS-PAGE were transferred to PVDF membrane for western blot detection with anti-Cry4Aa IgG.

Techniques Used: Activation Assay, Modification, Purification, SDS Page, Western Blot

11) Product Images from "NF-?B1 p105 Negatively Regulates TPL-2 MEK Kinase Activity"

Article Title: NF-?B1 p105 Negatively Regulates TPL-2 MEK Kinase Activity

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.14.4739-4752.2003

Both TPL-2 binding sites on p105 are required for optimal association with TPL-2. (A) Schematic diagram of HA-p105 mutants. (B) 293 cells were transfected with vectors encoding wild-type or deletion mutant forms of HA-p105. Biotinylated TPL-2 398-467 peptide, coupled to streptavidin-agarose beads, was then used to affinity purify HA-p105 from cell lysates. Isolated protein was detected by Western blotting. (C) 293 cells were cotransfected with vectors encoding TPL-2 and the indicated HA-p105 mutants or EV. In this experiment, to assay the interaction between TPL-2 and HA-p105 under more stringent conditions, cells were lysed and immunoprecipitated in radioimmunoprecipitation assay buffer. Anti-HA immunoprecipitates and cell lysates were Western blotted and probed sequentially with the indicated antibodies. The amount of TPL-2 epression vector was adjusted so that similar steady-state levels of protein expression in cell lysates were obtained with or without HA-p105.
Figure Legend Snippet: Both TPL-2 binding sites on p105 are required for optimal association with TPL-2. (A) Schematic diagram of HA-p105 mutants. (B) 293 cells were transfected with vectors encoding wild-type or deletion mutant forms of HA-p105. Biotinylated TPL-2 398-467 peptide, coupled to streptavidin-agarose beads, was then used to affinity purify HA-p105 from cell lysates. Isolated protein was detected by Western blotting. (C) 293 cells were cotransfected with vectors encoding TPL-2 and the indicated HA-p105 mutants or EV. In this experiment, to assay the interaction between TPL-2 and HA-p105 under more stringent conditions, cells were lysed and immunoprecipitated in radioimmunoprecipitation assay buffer. Anti-HA immunoprecipitates and cell lysates were Western blotted and probed sequentially with the indicated antibodies. The amount of TPL-2 epression vector was adjusted so that similar steady-state levels of protein expression in cell lysates were obtained with or without HA-p105.

Techniques Used: Binding Assay, Transfection, Mutagenesis, Isolation, Western Blot, Immunoprecipitation, Radio Immunoprecipitation, Plasmid Preparation, Expressing

The p105 DD is a binding site for TPL-2. (A) Schematic diagram of HA-p105 mutants. (B) 293 cells were cotransfected with TPL-2ΔC and the indicated HA-p105 mutants. Anti-HA MAb immunoprecipitates and cell lysates were Western blotted and probed with the indicated antibodies. The amounts of vector encoding TPL-2ΔC were adjusted to achieve equal protein expression in lysates with or without HA-p105. (C) TPL-2 was synthesized and labeled with [ 35 S]methionine-[ 35 S]cysteine by in vitro cell-free translation. Pulldowns were then performed with GST-p105 808-892 or GST control. Isolated protein was resolved by SDS-PAGE and revealed by fluorography.
Figure Legend Snippet: The p105 DD is a binding site for TPL-2. (A) Schematic diagram of HA-p105 mutants. (B) 293 cells were cotransfected with TPL-2ΔC and the indicated HA-p105 mutants. Anti-HA MAb immunoprecipitates and cell lysates were Western blotted and probed with the indicated antibodies. The amounts of vector encoding TPL-2ΔC were adjusted to achieve equal protein expression in lysates with or without HA-p105. (C) TPL-2 was synthesized and labeled with [ 35 S]methionine-[ 35 S]cysteine by in vitro cell-free translation. Pulldowns were then performed with GST-p105 808-892 or GST control. Isolated protein was resolved by SDS-PAGE and revealed by fluorography.

Techniques Used: Binding Assay, Western Blot, Plasmid Preparation, Expressing, Synthesized, Labeling, In Vitro, Isolation, SDS Page

p105 blocks interaction between TPL-2 and MEK. (A) 3T3 fibroblasts were transfected with EV or TPL-2 vector or in combination with HA-p105 vector or EV. Cell lysates were Western blotted and probed sequentially with the indicated antibodies. (B) 293 cells were transfected with expression vectors encoding Myc-TPL-2 with or without wild-type or mutant version of HA-p105. GST-MEK1(K207A) protein, bound to GSH-Sepharose 4B beads, was used to affinity purify Myc-TPL-2 from cell lysates. Isolated protein and protein expression in lysates was assayed by Western blotting. (C) Lysates from 293 cells transfected with Myc-TPL-2 were incubated with the indicated concentrations of p105 DD protein prior to addition of GST-MEK1(K207A) affinity ligand. Pulldowns and lysates were Western blotted.
Figure Legend Snippet: p105 blocks interaction between TPL-2 and MEK. (A) 3T3 fibroblasts were transfected with EV or TPL-2 vector or in combination with HA-p105 vector or EV. Cell lysates were Western blotted and probed sequentially with the indicated antibodies. (B) 293 cells were transfected with expression vectors encoding Myc-TPL-2 with or without wild-type or mutant version of HA-p105. GST-MEK1(K207A) protein, bound to GSH-Sepharose 4B beads, was used to affinity purify Myc-TPL-2 from cell lysates. Isolated protein and protein expression in lysates was assayed by Western blotting. (C) Lysates from 293 cells transfected with Myc-TPL-2 were incubated with the indicated concentrations of p105 DD protein prior to addition of GST-MEK1(K207A) affinity ligand. Pulldowns and lysates were Western blotted.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Expressing, Mutagenesis, Isolation, Incubation

). (C and D) 293 cells were cotransfected with vectors encoding the indicated Myc-TPL-2 or TPL-2 mutants and HA-p105 or EV(−). Anti-HA immunoprecipitates and cell lysates were sequentially immunoblotted with the indicated antibodies.
Figure Legend Snippet: ). (C and D) 293 cells were cotransfected with vectors encoding the indicated Myc-TPL-2 or TPL-2 mutants and HA-p105 or EV(−). Anti-HA immunoprecipitates and cell lysates were sequentially immunoblotted with the indicated antibodies.

Techniques Used:

Characterization of the interaction of the TPL-2 C terminus with p105. (A) Schematic diagram of recombinant p105 proteins. (B) Recombinant p105 497-968 protein was digested with trypsin with or without TPL-2 398-497 peptide for 60 min, resolved by 10% Bis-Tris gel electrophoresis, and Western blotted. Protein fragments were visualized by Coomassie brilliant blue staining. The mobilities of p105 497-968 and the fragments corresponding to p105 497-683 and p105 534-683 are indicated. (C) Surface plasmon resonance analysis of the interaction of p105 497-805 and p105 540-801 protein with biotinylated TPL-2 398-497 peptide immobilized on a streptavidin-coated sensor surface. An open arrow denotes the injection of biotinylated TPL-2 C-terminal peptide, closed arrow denotes the injection of indicated p105 protein. (D) Binding affinities of TPL-2 398-497 peptide for the indicated recombinant p105 proteins, as determined by surface plasmon resonance.
Figure Legend Snippet: Characterization of the interaction of the TPL-2 C terminus with p105. (A) Schematic diagram of recombinant p105 proteins. (B) Recombinant p105 497-968 protein was digested with trypsin with or without TPL-2 398-497 peptide for 60 min, resolved by 10% Bis-Tris gel electrophoresis, and Western blotted. Protein fragments were visualized by Coomassie brilliant blue staining. The mobilities of p105 497-968 and the fragments corresponding to p105 497-683 and p105 534-683 are indicated. (C) Surface plasmon resonance analysis of the interaction of p105 497-805 and p105 540-801 protein with biotinylated TPL-2 398-497 peptide immobilized on a streptavidin-coated sensor surface. An open arrow denotes the injection of biotinylated TPL-2 C-terminal peptide, closed arrow denotes the injection of indicated p105 protein. (D) Binding affinities of TPL-2 398-497 peptide for the indicated recombinant p105 proteins, as determined by surface plasmon resonance.

Techniques Used: Recombinant, Nucleic Acid Electrophoresis, Western Blot, Staining, SPR Assay, Injection, Binding Assay

p105 amino acids 497 to 539 are required for dimerization of the C-terminal half of p105. (A) Binding of TPL2 398-497 peptide to p105 497-805 protein was determined by ITC. The right panel shows integrated heat changes (▪), corrected for the heat of dilution, which were fitted by using a single binding site model ( K d = 58 nM; stoichiometry, n = 0.57). Under the same conditions, p105 540-801 protein did not bind TPL2 398-497 peptide (○). The left panel shows the raw data of the p105 497-805 titration in which the heat change of this endothermic binding reaction was measured in microcalories/second. (B) Molecular masses of recombinant p105 proteins were determined by sedimentation equilibrium ultracentrifugation. The lower panels show the protein distribution at equilibrium. The upper panels give the residuals in relation to the radial position. Experimental and theoretical molecular masses of the indicated recombinant p105 proteins are shown.
Figure Legend Snippet: p105 amino acids 497 to 539 are required for dimerization of the C-terminal half of p105. (A) Binding of TPL2 398-497 peptide to p105 497-805 protein was determined by ITC. The right panel shows integrated heat changes (▪), corrected for the heat of dilution, which were fitted by using a single binding site model ( K d = 58 nM; stoichiometry, n = 0.57). Under the same conditions, p105 540-801 protein did not bind TPL2 398-497 peptide (○). The left panel shows the raw data of the p105 497-805 titration in which the heat change of this endothermic binding reaction was measured in microcalories/second. (B) Molecular masses of recombinant p105 proteins were determined by sedimentation equilibrium ultracentrifugation. The lower panels show the protein distribution at equilibrium. The upper panels give the residuals in relation to the radial position. Experimental and theoretical molecular masses of the indicated recombinant p105 proteins are shown.

Techniques Used: Binding Assay, Titration, Recombinant, Sedimentation

TPL-2 MEK kinase activity is inhibited by p105 in vitro. (A and B) Myc-TPL-2 was isolated by immunoprecipitation from lysates of transfected 293 cells and then preincubated with the different amounts of the indicated recombinant p105 proteins or control buffer (−). In vitro kinase assays (KAs) were performed with GST-MEK1(K207A) as a substrate and phosphorylation determined by Western blotting of reaction mixtures and probing with an anti-phospho-MEK1/2 Ser217/Ser221 antibody. Equal loading of GST-MEK1(K207A) protein was confirmed by reprobing blots with anti-MEK1/2 antibody. Western blotting of anti-Myc immunoprecipitates (Ip) demonstrated that similar amounts of TPL-2 were assayed in each reaction (lower panel). (C) MEK1 phosphorylation in replicates of the experiments shown in A and B was quantified by laser densitometry ( n = 3). Data are presented as percentages of control MEK kinase activity. (D) Myc-Raf1(CAAX) was immunoprecipitated from lysates of transfected 293 cells and MEK kinase activity assayed as in panel A. Recombinant p105 protein was added to a final concentration of 5 μM.
Figure Legend Snippet: TPL-2 MEK kinase activity is inhibited by p105 in vitro. (A and B) Myc-TPL-2 was isolated by immunoprecipitation from lysates of transfected 293 cells and then preincubated with the different amounts of the indicated recombinant p105 proteins or control buffer (−). In vitro kinase assays (KAs) were performed with GST-MEK1(K207A) as a substrate and phosphorylation determined by Western blotting of reaction mixtures and probing with an anti-phospho-MEK1/2 Ser217/Ser221 antibody. Equal loading of GST-MEK1(K207A) protein was confirmed by reprobing blots with anti-MEK1/2 antibody. Western blotting of anti-Myc immunoprecipitates (Ip) demonstrated that similar amounts of TPL-2 were assayed in each reaction (lower panel). (C) MEK1 phosphorylation in replicates of the experiments shown in A and B was quantified by laser densitometry ( n = 3). Data are presented as percentages of control MEK kinase activity. (D) Myc-Raf1(CAAX) was immunoprecipitated from lysates of transfected 293 cells and MEK kinase activity assayed as in panel A. Recombinant p105 protein was added to a final concentration of 5 μM.

Techniques Used: Activity Assay, In Vitro, Isolation, Immunoprecipitation, Transfection, Recombinant, Western Blot, Concentration Assay

12) Product Images from "TPD52 expression increases neutral lipid storage within cultured cells"

Article Title: TPD52 expression increases neutral lipid storage within cultured cells

Journal: Journal of Cell Science

doi: 10.1242/jcs.167692

Direct interactions between TPD52 and ADRP or TIP47. (A) Analyses of direct interactions between TPD52 or TPD52L1 and ADRP or TIP47, along with known positive and negative controls using the yeast two-hybrid system. (+), growth on solid SD/−His−Leu−Trp
Figure Legend Snippet: Direct interactions between TPD52 and ADRP or TIP47. (A) Analyses of direct interactions between TPD52 or TPD52L1 and ADRP or TIP47, along with known positive and negative controls using the yeast two-hybrid system. (+), growth on solid SD/−His−Leu−Trp

Techniques Used:

Colocalisation of ADRP and TPD52 in untreated and oleic-acid-treated D52-2-7 cells. . Cells were colabelled with TPD52 (green) and ADRP (pseudo-coloured, red). Enlarged images of the regions indicated
Figure Legend Snippet: Colocalisation of ADRP and TPD52 in untreated and oleic-acid-treated D52-2-7 cells. . Cells were colabelled with TPD52 (green) and ADRP (pseudo-coloured, red). Enlarged images of the regions indicated

Techniques Used:

Colocalisation of TPD52 and the Golgi marker Gm130 in untreated and oleic-acid-treated D52-2-7 cells. Cells were untreated (A) or supplemented with 400 μM oleic acid in fatty-acid-free BSA (OA) for 6 h (B), fixed and subjected
Figure Legend Snippet: Colocalisation of TPD52 and the Golgi marker Gm130 in untreated and oleic-acid-treated D52-2-7 cells. Cells were untreated (A) or supplemented with 400 μM oleic acid in fatty-acid-free BSA (OA) for 6 h (B), fixed and subjected

Techniques Used: Marker

Increased lipid storage in TPD52- but not TPD52L1-expressing 3T3 cells. (A) Alignment of TPD52 (Uniprot identifier P55327-2) and TPD52L1 (Uniprot identifier Q16890-1) sequences, shown using the one-letter code, produced by the EMBOSS Needle algorithm.
Figure Legend Snippet: Increased lipid storage in TPD52- but not TPD52L1-expressing 3T3 cells. (A) Alignment of TPD52 (Uniprot identifier P55327-2) and TPD52L1 (Uniprot identifier Q16890-1) sequences, shown using the one-letter code, produced by the EMBOSS Needle algorithm.

Techniques Used: Expressing, Produced

TPD52-expressing cells store more lipids upon oleic acid supplementation. (A) Indirect immunofluorescence analyses of control (3T3-parent, vector-3), and TPD52-expressing (D52-1-12, D52-2-1, D52-2-7) 3T3 cells with (right panels, 24 h) or without
Figure Legend Snippet: TPD52-expressing cells store more lipids upon oleic acid supplementation. (A) Indirect immunofluorescence analyses of control (3T3-parent, vector-3), and TPD52-expressing (D52-1-12, D52-2-1, D52-2-7) 3T3 cells with (right panels, 24 h) or without

Techniques Used: Expressing, Immunofluorescence, Plasmid Preparation

Altered fatty acid metabolism in TPD52-expressing 3T3 cells. (A) Triglyceride levels measured in parent, vector and TPD52-expressing 3T3 cells. Incorporation rates of (B) newly-synthesised free fatty acid (from [ 3 H]acetate) into TAG, as a measure of
Figure Legend Snippet: Altered fatty acid metabolism in TPD52-expressing 3T3 cells. (A) Triglyceride levels measured in parent, vector and TPD52-expressing 3T3 cells. Incorporation rates of (B) newly-synthesised free fatty acid (from [ 3 H]acetate) into TAG, as a measure of

Techniques Used: Expressing, Plasmid Preparation

Colocalisation of Gm130 or TPD52 with ARL1 and ADRP in untreated and oleic-acid-treated D52-2-7 cells. Cells were untreated or supplemented with 400 μM in fatty-acid-free BSA (OA) for 24 h, fixed, and subjected to indirect immunofluorescence
Figure Legend Snippet: Colocalisation of Gm130 or TPD52 with ARL1 and ADRP in untreated and oleic-acid-treated D52-2-7 cells. Cells were untreated or supplemented with 400 μM in fatty-acid-free BSA (OA) for 24 h, fixed, and subjected to indirect immunofluorescence

Techniques Used: Immunofluorescence

13) Product Images from "The Novel Zinc Finger Protein dASCIZ Regulates Mitosis in Drosophila via an Essential Role in Dynein Light-Chain Expression"

Article Title: The Novel Zinc Finger Protein dASCIZ Regulates Mitosis in Drosophila via an Essential Role in Dynein Light-Chain Expression

Journal: Genetics

doi: 10.1534/genetics.113.159541

dASCIZ and Ctp are required for progression through late stages of mitosis. (A) Proportion of cells in wing imaginal discs at the indicated mitotic stages based on cell morphology and PH3 staining. Compared with the control, wings expressing dASCIZ RNAi
Figure Legend Snippet: dASCIZ and Ctp are required for progression through late stages of mitosis. (A) Proportion of cells in wing imaginal discs at the indicated mitotic stages based on cell morphology and PH3 staining. Compared with the control, wings expressing dASCIZ RNAi

Techniques Used: Staining, Expressing

The dASCIZ RNAi wing phenotype is phenocopied by Ctp knockdown and rescued by Ctp overexpression. (A–E) Representative images of male adult wings with Engrailed–GAL4 (En) driver. (A) Control En–GAL4/ +. (B) En > UAS–Ctp
Figure Legend Snippet: The dASCIZ RNAi wing phenotype is phenocopied by Ctp knockdown and rescued by Ctp overexpression. (A–E) Representative images of male adult wings with Engrailed–GAL4 (En) driver. (A) Control En–GAL4/ +. (B) En > UAS–Ctp

Techniques Used: Over Expression

dASCIZ is required for Ctp expression. (A) qPCR for dASCIZ after global knockdown, fold change relative to control (error bars represent 95% CI, P = 0.0008 based on ΔΔCT value, normalized to actin). (B) Ctp mRNA levels are reduced in response
Figure Legend Snippet: dASCIZ is required for Ctp expression. (A) qPCR for dASCIZ after global knockdown, fold change relative to control (error bars represent 95% CI, P = 0.0008 based on ΔΔCT value, normalized to actin). (B) Ctp mRNA levels are reduced in response

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

dASCIZ knockdown results in increased mitotic index, which can be suppressed by Ctp co-overexpression. En–GAL4 expression in the PC of wing imaginal discs is marked with RFP (outlined in white) and mitotic cells are labeled with phospho-histone
Figure Legend Snippet: dASCIZ knockdown results in increased mitotic index, which can be suppressed by Ctp co-overexpression. En–GAL4 expression in the PC of wing imaginal discs is marked with RFP (outlined in white) and mitotic cells are labeled with phospho-histone

Techniques Used: Over Expression, Expressing, Labeling

Characterization of dASCIZ. (A) Schematic comparison of human and Drosophila ASCIZ; green lollipops indicate TQT motifs, and gray lollipops indicate other SQ or TQ motifs. (B) Pulldown of recombinant His 6 -tagged Ctp or human DYNLL1 with the GST-fused
Figure Legend Snippet: Characterization of dASCIZ. (A) Schematic comparison of human and Drosophila ASCIZ; green lollipops indicate TQT motifs, and gray lollipops indicate other SQ or TQ motifs. (B) Pulldown of recombinant His 6 -tagged Ctp or human DYNLL1 with the GST-fused

Techniques Used: Recombinant

dASCIZ is required for cell survival through Ctp. (A and B). Basal sections of wing imaginal discs, showing extruded centrosomin-positive mitotic cells in the disc expressing En > dASCIZ RNAi (B) but not in the control (A), costained for DNA with
Figure Legend Snippet: dASCIZ is required for cell survival through Ctp. (A and B). Basal sections of wing imaginal discs, showing extruded centrosomin-positive mitotic cells in the disc expressing En > dASCIZ RNAi (B) but not in the control (A), costained for DNA with

Techniques Used: Expressing

14) Product Images from "The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation"

Article Title: The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1002480

The structure of the TPR domain of Rrp5. (A) Cartoon representation of the crystal structure. (B) Surface charge distribution of the TPR domain of Rrp5. (C) Surface representation of amino acid conservation calculated using the ConSurf server [ 35 ] and displayed in PyMOL (The PyMOL Molecular Graphics System, Version 1.0r1 Schrödinger). The program ConSurf uses the provided sequence ( S . cerevisiae Rrp5) to find the 150 closest homologs (lowest E-values) for generating a multiple sequence alignment. The organisms used for Rrp5-TPR can be found here: http://consurf.tau.ac.il/results/1463756501/query_final_homolougs.html . The conserved patches on the surface of the TPR motif that interact with Rok1 are marked in red. (D) Mutations in conserved charged areas of the TPR domain are deleterious in vivo. TPR2: E1509K/E1510K/E1512K and TPR7: K1686E/K1689E. (E) Mutations in conserved charged areas of the TPR domain weaken the Rok1•Rrp5 interaction in vitro. (F) Position of the TPR mutations in the 3-D reconstruction of the Rrp5•Rok1 complex. (G) Recombinant GST-Rok1 immobilized on glutathione (GSH) sepharose resin interacts with recombinant, purified Rrp5 FL , Rrp5_C7, and Rrp5_C6, but not Rrp5_C5. (H) Maltose binding protein (MBP) or glutathione S-transferase (GST) alone does not bind to the Rrp5 fragments tested here. I, input; W, wash; E, elution.
Figure Legend Snippet: The structure of the TPR domain of Rrp5. (A) Cartoon representation of the crystal structure. (B) Surface charge distribution of the TPR domain of Rrp5. (C) Surface representation of amino acid conservation calculated using the ConSurf server [ 35 ] and displayed in PyMOL (The PyMOL Molecular Graphics System, Version 1.0r1 Schrödinger). The program ConSurf uses the provided sequence ( S . cerevisiae Rrp5) to find the 150 closest homologs (lowest E-values) for generating a multiple sequence alignment. The organisms used for Rrp5-TPR can be found here: http://consurf.tau.ac.il/results/1463756501/query_final_homolougs.html . The conserved patches on the surface of the TPR motif that interact with Rok1 are marked in red. (D) Mutations in conserved charged areas of the TPR domain are deleterious in vivo. TPR2: E1509K/E1510K/E1512K and TPR7: K1686E/K1689E. (E) Mutations in conserved charged areas of the TPR domain weaken the Rok1•Rrp5 interaction in vitro. (F) Position of the TPR mutations in the 3-D reconstruction of the Rrp5•Rok1 complex. (G) Recombinant GST-Rok1 immobilized on glutathione (GSH) sepharose resin interacts with recombinant, purified Rrp5 FL , Rrp5_C7, and Rrp5_C6, but not Rrp5_C5. (H) Maltose binding protein (MBP) or glutathione S-transferase (GST) alone does not bind to the Rrp5 fragments tested here. I, input; W, wash; E, elution.

Techniques Used: Sequencing, In Vivo, In Vitro, Recombinant, Purification, Binding Assay

Rrp5 binds to 40S ribosomal subunits in vivo and in vitro. (A) Ultracentrifugation pelleting experiments demonstrate that recombinant full-length Rrp5 binds purified mature 40S subunits in vitro. S, supernatant; P, pellet. (B) The three C-terminal S1 domains are essential for the interaction between Rrp5 and 40S subunits in vitro. Recombinant Rrp5 fragments are used in the same pelleting experiment as 1A. (C) Gradient centrifugation demonstrates a role for the three C-terminal S1 domains and S1–S5 for binding to preribosomes in vivo. (D) Gradient sedimentation experiments of various recombinant Rrp5•Rok1•AMPPCP complexes demonstrate that S1–S5 contributes to Rrp5 binding to 40S ribosomes in vitro. Note that the more sensitive gradient sedimentation experiments are required to demonstrate the more quantitative than qualitative differences in Rrp8_C8 and Rrp5_C7 binding. Because Rrp5_N9 and Rrp5_N12 do not bind Rok1, binding of these fragments requires the pelleting assay. All experiments were repeated at least twice, and representative data are shown.
Figure Legend Snippet: Rrp5 binds to 40S ribosomal subunits in vivo and in vitro. (A) Ultracentrifugation pelleting experiments demonstrate that recombinant full-length Rrp5 binds purified mature 40S subunits in vitro. S, supernatant; P, pellet. (B) The three C-terminal S1 domains are essential for the interaction between Rrp5 and 40S subunits in vitro. Recombinant Rrp5 fragments are used in the same pelleting experiment as 1A. (C) Gradient centrifugation demonstrates a role for the three C-terminal S1 domains and S1–S5 for binding to preribosomes in vivo. (D) Gradient sedimentation experiments of various recombinant Rrp5•Rok1•AMPPCP complexes demonstrate that S1–S5 contributes to Rrp5 binding to 40S ribosomes in vitro. Note that the more sensitive gradient sedimentation experiments are required to demonstrate the more quantitative than qualitative differences in Rrp8_C8 and Rrp5_C7 binding. Because Rrp5_N9 and Rrp5_N12 do not bind Rok1, binding of these fragments requires the pelleting assay. All experiments were repeated at least twice, and representative data are shown.

Techniques Used: In Vivo, In Vitro, Recombinant, Purification, Gradient Centrifugation, Binding Assay, Sedimentation

15) Product Images from "Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding"

Article Title: Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding

Journal: Virology Journal

doi: 10.1186/1743-422X-5-46

Binding of mutant HCV E1E2 glycoproteins to soluble CD81 . (A) 293T cells transfected with HCV E1E2 wt or mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with soluble CD81-GST fusion protein. Binding to CD81 was determined by Western Blot analysis of E2 and the GST tag. As a negative control, GST protein without soluble CD81 was incubated with HCV wt. Image is a composite. (B) 293T cells transfected with HCV E1E2 wt or specific mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with AR3A (C1) conformational antibody to assess conformation of mutations. Immunoprecipitated proteins were detected by subsequent Western Blot analysis of E2. Image is a composite.
Figure Legend Snippet: Binding of mutant HCV E1E2 glycoproteins to soluble CD81 . (A) 293T cells transfected with HCV E1E2 wt or mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with soluble CD81-GST fusion protein. Binding to CD81 was determined by Western Blot analysis of E2 and the GST tag. As a negative control, GST protein without soluble CD81 was incubated with HCV wt. Image is a composite. (B) 293T cells transfected with HCV E1E2 wt or specific mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with AR3A (C1) conformational antibody to assess conformation of mutations. Immunoprecipitated proteins were detected by subsequent Western Blot analysis of E2. Image is a composite.

Techniques Used: Binding Assay, Mutagenesis, Transfection, Expressing, Incubation, Protein Binding, Western Blot, Negative Control, Immunoprecipitation

16) Product Images from "XBP1 Promotes Triple Negative Breast Cancer By Controlling the HIF1 α Pathway"

Article Title: XBP1 Promotes Triple Negative Breast Cancer By Controlling the HIF1 α Pathway

Journal: Nature

doi: 10.1038/nature13119

Schema depicting the interaction of XBP1 and HIF1α in TNBC XBP1 and HIF1α cooperatively regulate HIF1α targets in TNBC. In the setting of a tumor microenvironment, hypoxia further induces XBP1 activation, active XBP1s in turn interacts with HIF1α to stimulate and augment the transactivation of HIF1 target genes that promote cancer progression.
Figure Legend Snippet: Schema depicting the interaction of XBP1 and HIF1α in TNBC XBP1 and HIF1α cooperatively regulate HIF1α targets in TNBC. In the setting of a tumor microenvironment, hypoxia further induces XBP1 activation, active XBP1s in turn interacts with HIF1α to stimulate and augment the transactivation of HIF1 target genes that promote cancer progression.

Techniques Used: Activation Assay

Overexpression of constitutively activated HIF1α rescues XBP1 knockdown phenotype a , Immunoblotting analysis of cell lysates of MDA-MB-231 cells infected with retrovirus encoding control vector or HA-HIF1α dPA was performed using anti-HA or anti-actin antibody. b , XBP1 splicing is not affected by HIF1α or HIF2α activation. RT-PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells expressing control vector, HA-HIF1α dPA or HA-HIF2α dPA. Expression of HA-HIF1α dPA is shown in ( a ) and expression of HIF2α is shown in the right panel. c , XBP1 splicing is not affected by HIF1α or HIF2α depletion. RT-PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells infected with control, shHIF1 α or shHIF2 α lentivirus. Knockdown efficiency of HIF1 α or HIF2 α is shown in the middle and right panels. d-e , Expression of constitutively activated HIF1α doesn't affect XBP1 expression ( d ), but restores HIF1α targets expression ( e ). RT-PCR analysis of XBP1 total ( d ), XBP1s ( d ) and VEGFA, PDK1, DDIT4 ( e ) in control shRNA (shCtrl), XBP1 shRNA ( shXBP1 ), or XBP1 shRNA plus constitutively activated HIF1α (shXBP1+HIF1α dPA) infected MDA-MB-231 cells. Data ( b-e ) are shown as mean ± SD of technical triplicates. *p
Figure Legend Snippet: Overexpression of constitutively activated HIF1α rescues XBP1 knockdown phenotype a , Immunoblotting analysis of cell lysates of MDA-MB-231 cells infected with retrovirus encoding control vector or HA-HIF1α dPA was performed using anti-HA or anti-actin antibody. b , XBP1 splicing is not affected by HIF1α or HIF2α activation. RT-PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells expressing control vector, HA-HIF1α dPA or HA-HIF2α dPA. Expression of HA-HIF1α dPA is shown in ( a ) and expression of HIF2α is shown in the right panel. c , XBP1 splicing is not affected by HIF1α or HIF2α depletion. RT-PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells infected with control, shHIF1 α or shHIF2 α lentivirus. Knockdown efficiency of HIF1 α or HIF2 α is shown in the middle and right panels. d-e , Expression of constitutively activated HIF1α doesn't affect XBP1 expression ( d ), but restores HIF1α targets expression ( e ). RT-PCR analysis of XBP1 total ( d ), XBP1s ( d ) and VEGFA, PDK1, DDIT4 ( e ) in control shRNA (shCtrl), XBP1 shRNA ( shXBP1 ), or XBP1 shRNA plus constitutively activated HIF1α (shXBP1+HIF1α dPA) infected MDA-MB-231 cells. Data ( b-e ) are shown as mean ± SD of technical triplicates. *p

Techniques Used: Over Expression, Multiple Displacement Amplification, Infection, Plasmid Preparation, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, shRNA

Role of XBP1 in luminal breast cancer a , XBP1 splicing is induced by hypoxia and glucose deprivation in luminal cancer cells. RT-PCR of XBP1 splicing in T47D and SKBR3 cells under different treatments for 24h. XBP1u: unspliced XBP1, XBP1s: spliced XBP1. Hypoxia: 0.1% O 2 . b . Left panel: Quantification of soft agar colony formation in untreated and control shRNA or XBP1 shRNA infected breast cancer cells. Experiments were performed in triplicate and data are shown as mean ± SD. **p
Figure Legend Snippet: Role of XBP1 in luminal breast cancer a , XBP1 splicing is induced by hypoxia and glucose deprivation in luminal cancer cells. RT-PCR of XBP1 splicing in T47D and SKBR3 cells under different treatments for 24h. XBP1u: unspliced XBP1, XBP1s: spliced XBP1. Hypoxia: 0.1% O 2 . b . Left panel: Quantification of soft agar colony formation in untreated and control shRNA or XBP1 shRNA infected breast cancer cells. Experiments were performed in triplicate and data are shown as mean ± SD. **p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, shRNA, Infection

XBP1 and HIF1α co-occupy HIF1α targets a , Track view of XBP1 ChIP-seq density profile (two biological replicates) on HIF1α target genes. b , XBP1 and HIF1α co-bind to DDIT4 , VEGFA , and PDK1 promoters under hypoxic conditions. A ChIP assay was performed using anti-XBP1 or anti-HIF1α antibody to detect enriched fragments. GST antibody was used as mock ChIP control. c , XBP1 and HIF1α co-bind to JMJD1A and JMJD2C promoters under hypoxic conditions. Upper panel: Schematic diagram of the primer locations across the JMJD2C or JMJD1A promoter. Grey boxes indicate exon. A ChIP assay was performed using anti-XBP1 polyclonal antibody or anti-HIF1α polyclonal antibody to detect enriched fragments. Fold enrichment is the relative abundance of DNA fragments at the amplified region over a control amplified region. GST antibody was used as mock ChIP control. d , XBP1s and HIF1α co-occupy JMJD1A , DDIT4 , NDRG1 , PDK1 and VEGFA promoters. A ChIP-re-ChIP assay was performed using the anti-XBP1s antibody first (X). The eluants were then subjected to a second ChIP assay using an anti-HIF1α antibody (XH) or a control IgG antibody (XC). All ChIP Data ( b-d ) are shown as mean ± SD of technical triplicates. Results show a representative of two independent experiments. *p
Figure Legend Snippet: XBP1 and HIF1α co-occupy HIF1α targets a , Track view of XBP1 ChIP-seq density profile (two biological replicates) on HIF1α target genes. b , XBP1 and HIF1α co-bind to DDIT4 , VEGFA , and PDK1 promoters under hypoxic conditions. A ChIP assay was performed using anti-XBP1 or anti-HIF1α antibody to detect enriched fragments. GST antibody was used as mock ChIP control. c , XBP1 and HIF1α co-bind to JMJD1A and JMJD2C promoters under hypoxic conditions. Upper panel: Schematic diagram of the primer locations across the JMJD2C or JMJD1A promoter. Grey boxes indicate exon. A ChIP assay was performed using anti-XBP1 polyclonal antibody or anti-HIF1α polyclonal antibody to detect enriched fragments. Fold enrichment is the relative abundance of DNA fragments at the amplified region over a control amplified region. GST antibody was used as mock ChIP control. d , XBP1s and HIF1α co-occupy JMJD1A , DDIT4 , NDRG1 , PDK1 and VEGFA promoters. A ChIP-re-ChIP assay was performed using the anti-XBP1s antibody first (X). The eluants were then subjected to a second ChIP assay using an anti-HIF1α antibody (XH) or a control IgG antibody (XC). All ChIP Data ( b-d ) are shown as mean ± SD of technical triplicates. Results show a representative of two independent experiments. *p

Techniques Used: Chromatin Immunoprecipitation, Amplification

XBP1 silencing blocks TNBC cell growth and invasiveness a-b, RT-PCR analysis of XBP1 splicing in luminal and basal-like cell lines ( a ) or primary tissues from 6 TNBC patients and 5 ER/PR+ patients ( b ). XBP1u: unspliced XBP1, XBP1s: spliced XBP1. β-actin was used as loading control. c, Representative bioluminescent images of orthotopic tumors formed by MDA-MB-231 cells as in ( Extended Data 1d ). Bioluminescent images were obtained 5 days after transplantation and serially after mice were begun on chow containing doxycycline (day 19) for 8 weeks. Pictures shown are the day19 image (Before Dox) and day 64 image (After Dox). d, Quantification of imaging studies as in ( c ). Data are shown as mean ± SD of biological replicates (n=8). *p
Figure Legend Snippet: XBP1 silencing blocks TNBC cell growth and invasiveness a-b, RT-PCR analysis of XBP1 splicing in luminal and basal-like cell lines ( a ) or primary tissues from 6 TNBC patients and 5 ER/PR+ patients ( b ). XBP1u: unspliced XBP1, XBP1s: spliced XBP1. β-actin was used as loading control. c, Representative bioluminescent images of orthotopic tumors formed by MDA-MB-231 cells as in ( Extended Data 1d ). Bioluminescent images were obtained 5 days after transplantation and serially after mice were begun on chow containing doxycycline (day 19) for 8 weeks. Pictures shown are the day19 image (Before Dox) and day 64 image (After Dox). d, Quantification of imaging studies as in ( c ). Data are shown as mean ± SD of biological replicates (n=8). *p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Transplantation Assay, Mouse Assay, Imaging

17) Product Images from "A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid"

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

Journal: Scientific Reports

doi: 10.1038/s41598-019-41426-4

mOH is present in immature mouse ULF. ( A ) The immature mouse ULF and the gelatinolytic enzyme complex were separated by non-reducing SDS-PAGE and then analyzed by Western blotting, probed for mOH by POHA. The POHA was purified from rabbit anti-mOH sera. Lane 1: immature mouse ULF (5 μl). Lane 2: the gelatinolytic enzyme complex (0.5 μg) that was the resulting product after immature mouse ULF was purified by gel filtration and DEAE-Sepharose chromatography. ( B ) The immature mouse ULF and the gelatinolytic enzyme complex were separated by reducing SDS-PAGE and then analyzed by Western blotting, probed for reducing mOH by rabbit anti-GST-mOH[870–940] sera. Lane 1: immature mouse ULF (5 μl). Lane 2: the gelatinolytic enzyme complex (0.5 μg).
Figure Legend Snippet: mOH is present in immature mouse ULF. ( A ) The immature mouse ULF and the gelatinolytic enzyme complex were separated by non-reducing SDS-PAGE and then analyzed by Western blotting, probed for mOH by POHA. The POHA was purified from rabbit anti-mOH sera. Lane 1: immature mouse ULF (5 μl). Lane 2: the gelatinolytic enzyme complex (0.5 μg) that was the resulting product after immature mouse ULF was purified by gel filtration and DEAE-Sepharose chromatography. ( B ) The immature mouse ULF and the gelatinolytic enzyme complex were separated by reducing SDS-PAGE and then analyzed by Western blotting, probed for reducing mOH by rabbit anti-GST-mOH[870–940] sera. Lane 1: immature mouse ULF (5 μl). Lane 2: the gelatinolytic enzyme complex (0.5 μg).

Techniques Used: SDS Page, Western Blot, Purification, Filtration, Chromatography

Purification of the ULF gelatinolytic enzyme. ( A ) Gelatin zymogram of immature mouse ULF. The ULF (6 μl) was analyzed by gelatin zymography. ( B ) Elution profile of ULF gelatinolytic enzyme carried out by gel filtration chromatography. Soluble ULF (1 ml) was subjected to gel filtration chromatography on a Sephacryl S-400 column. The elution was monitored by UV spectrophotometry with absorbance at 280 nm (—). The gelatinolytic activity of 26 kDa enzyme in each indicated fraction was quantitatively detected (---). Arrows indicate the protein markers eluted from the same column. The protein markers are human α 2 MG (725 kDa), apoferritin (443 kDa) and carbonic anhydrase (29 kDa). The horizontal bar represents the pooled fractions for further purification by the DEAE-Sepharose chromatography. ( C ) Elution profile of DEAE-Sepharose chromatography. The pooled fractions are marked by a horizontal bar. ( D ) Gelatin zymographic assay. Lane 1: immature mouse ULF (6 μl). Lane 2: the pooled fractions (0.3 ml) from gel filtration. Line 3: the pooled fractions (0.25 ml) from DEAE-Sepharose chromatography.
Figure Legend Snippet: Purification of the ULF gelatinolytic enzyme. ( A ) Gelatin zymogram of immature mouse ULF. The ULF (6 μl) was analyzed by gelatin zymography. ( B ) Elution profile of ULF gelatinolytic enzyme carried out by gel filtration chromatography. Soluble ULF (1 ml) was subjected to gel filtration chromatography on a Sephacryl S-400 column. The elution was monitored by UV spectrophotometry with absorbance at 280 nm (—). The gelatinolytic activity of 26 kDa enzyme in each indicated fraction was quantitatively detected (---). Arrows indicate the protein markers eluted from the same column. The protein markers are human α 2 MG (725 kDa), apoferritin (443 kDa) and carbonic anhydrase (29 kDa). The horizontal bar represents the pooled fractions for further purification by the DEAE-Sepharose chromatography. ( C ) Elution profile of DEAE-Sepharose chromatography. The pooled fractions are marked by a horizontal bar. ( D ) Gelatin zymographic assay. Lane 1: immature mouse ULF (6 μl). Lane 2: the pooled fractions (0.3 ml) from gel filtration. Line 3: the pooled fractions (0.25 ml) from DEAE-Sepharose chromatography.

Techniques Used: Purification, Zymography, Filtration, Chromatography, Spectrophotometry, Activity Assay

18) Product Images from "Tfg3, a subunit of the general transcription factor TFIIF in Schizosaccharomyces pombe, functions under stress conditions"

Article Title: Tfg3, a subunit of the general transcription factor TFIIF in Schizosaccharomyces pombe, functions under stress conditions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh1000

Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with GSH–Sepharose. Proteins bound to the resin were analyzed by SDS–PAGE and
Figure Legend Snippet: Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with GSH–Sepharose. Proteins bound to the resin were analyzed by SDS–PAGE and

Techniques Used: Incubation, SDS Page

19) Product Images from "Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein"

Article Title: Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-2569-8_28

GSH bead-based flow cytometry assay configurations for quantitative measurements of GTPase-effector protein binding. ( a ) Assay design for detecting Rab7 and Rab-interacting lysosomal protein (RILP) effector protein interaction based on detection of bound fluorescent BODIPY-GTP. GST-RILP (Rab binding domain of RILP only) is immobilized on 13 μm Superdex beads coated with GSH and incubated with purified His-tagged Rab7 complexed to fluorescent BODIPY-GTP. Flow cytometry detection is based on bead-associated fluorescence when His-Rab7-BODIPY-GTP binds to RILP. ( b ) Assay design for detecting Rab7 and Rab-interacting lysosomal protein (RILP) protein interaction based on detection of GFP-tagged Rab7. GST-RILP (Rab-binding domain of RILP only) is immobilized on 13 μm Superdex beads coated with GSH and incubated with GFP- tagged Rab7 complexed to nonhydrolyzable GTP-γ-S. Flow cytometry detection is based on bead-associated fluorescence when GFP-Rab7-GTP-γ-S binds to RILP
Figure Legend Snippet: GSH bead-based flow cytometry assay configurations for quantitative measurements of GTPase-effector protein binding. ( a ) Assay design for detecting Rab7 and Rab-interacting lysosomal protein (RILP) effector protein interaction based on detection of bound fluorescent BODIPY-GTP. GST-RILP (Rab binding domain of RILP only) is immobilized on 13 μm Superdex beads coated with GSH and incubated with purified His-tagged Rab7 complexed to fluorescent BODIPY-GTP. Flow cytometry detection is based on bead-associated fluorescence when His-Rab7-BODIPY-GTP binds to RILP. ( b ) Assay design for detecting Rab7 and Rab-interacting lysosomal protein (RILP) protein interaction based on detection of GFP-tagged Rab7. GST-RILP (Rab-binding domain of RILP only) is immobilized on 13 μm Superdex beads coated with GSH and incubated with GFP- tagged Rab7 complexed to nonhydrolyzable GTP-γ-S. Flow cytometry detection is based on bead-associated fluorescence when GFP-Rab7-GTP-γ-S binds to RILP

Techniques Used: Flow Cytometry, Cytometry, Protein Binding, Binding Assay, Incubation, Purification, Fluorescence

GSH bead-based flow cytometry assays for quantitative measurements of Rab7 guanine nucleotide binding and dissociation kinetics. ( a ) Assay design for detecting nucleotide binding and dissociation kinetics on Rab7 based on detection of bound fluorescent BODIPY-GTP. GST-Rab7 is immobilized on 13 μm Superdex beads coated with GSH and detection is based on fluorescent BODIPY-GTP binding. ( b ) BODIPY-GTP (100 nM final) was added to GST-Rab7 immobilized on GSH beads suspended in 300 μl of buffer ( first arrow ). The ligand was allowed to bind for 100 s and then DMSO (1 % final) or CID 1067700 (10 μM final) was added at 150 s ( second arrow ). While the addition of a competitive guanine nucleotide-binding inhibitor (CID 1067700) causes dissociation of BODIPY-GTP, addition of DMSO has no effect on BODIPY-GTP-binding kinetics
Figure Legend Snippet: GSH bead-based flow cytometry assays for quantitative measurements of Rab7 guanine nucleotide binding and dissociation kinetics. ( a ) Assay design for detecting nucleotide binding and dissociation kinetics on Rab7 based on detection of bound fluorescent BODIPY-GTP. GST-Rab7 is immobilized on 13 μm Superdex beads coated with GSH and detection is based on fluorescent BODIPY-GTP binding. ( b ) BODIPY-GTP (100 nM final) was added to GST-Rab7 immobilized on GSH beads suspended in 300 μl of buffer ( first arrow ). The ligand was allowed to bind for 100 s and then DMSO (1 % final) or CID 1067700 (10 μM final) was added at 150 s ( second arrow ). While the addition of a competitive guanine nucleotide-binding inhibitor (CID 1067700) causes dissociation of BODIPY-GTP, addition of DMSO has no effect on BODIPY-GTP-binding kinetics

Techniques Used: Flow Cytometry, Cytometry, Binding Assay

20) Product Images from "Prolyl Hydroxylase Domain Protein 2 (PHD2) Binds a Pro-Xaa-Leu-Glu Motif, Linking It to the Heat Shock Protein 90 Pathway *"

Article Title: Prolyl Hydroxylase Domain Protein 2 (PHD2) Binds a Pro-Xaa-Leu-Glu Motif, Linking It to the Heat Shock Protein 90 Pathway *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.440552

The PHD2-p23 interaction facilitates prolyl hydroxylation of HIF-1α. A , HeLa cells were treated with control ( Con ) or p23 siRNA, and cellular extracts were prepared. GST or GST-HIF-1α (531–575) prebound to GSH-agarose was incubated
Figure Legend Snippet: The PHD2-p23 interaction facilitates prolyl hydroxylation of HIF-1α. A , HeLa cells were treated with control ( Con ) or p23 siRNA, and cellular extracts were prepared. GST or GST-HIF-1α (531–575) prebound to GSH-agarose was incubated

Techniques Used: Incubation

21) Product Images from "Calmodulin in complex with the first IQ motif of myosin-5a functions as an intact calcium sensor"

Article Title: Calmodulin in complex with the first IQ motif of myosin-5a functions as an intact calcium sensor

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

doi: 10.1073/pnas.1607702113

Myo5a-HMM and the MD-IQ1 bind to GST-GTD in a Ca 2+ -dependent manner. FLAG-tagged MD-IQ1 or Myo5a-HMM was incubated with GST-GTD or GST and was pulled down by GSH-Sepharose under EGTA or pCa4 conditions. The pulled-down samples (20 μL each) were
Figure Legend Snippet: Myo5a-HMM and the MD-IQ1 bind to GST-GTD in a Ca 2+ -dependent manner. FLAG-tagged MD-IQ1 or Myo5a-HMM was incubated with GST-GTD or GST and was pulled down by GSH-Sepharose under EGTA or pCa4 conditions. The pulled-down samples (20 μL each) were

Techniques Used: Incubation

22) Product Images from "Rice DWARF14 acts as an unconventional hormone receptor for strigolactone"

Article Title: Rice DWARF14 acts as an unconventional hormone receptor for strigolactone

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/ery014

Rice D14 physically interacts with the Arabidopsis SL signaling components. (A) Rice D14 efficiently bound Arabidopsis MAX2 in the presence of rac -GR24. Pull-down assay using recombinant His 6 -MAX2 and GST–D14 or GST–AtD14 in the absence or presence of rac -GR24. GST-fused proteins were detected by anti-GST antibody and the PVDF membrane was stained with MemStain to show equal loading. (B) Rice D14 efficiently bound Arabidopsis SMXL6 in the presence of rac -GR24. Pull-down assay using recombinant Flag-SMXL6 and GST–D14 or GST–AtD14 in the absence or presence of rac -GR24. GST-fused proteins were detected by anti-GST antibody and the PVDF membrane was stained with MemStain to show equal loading.
Figure Legend Snippet: Rice D14 physically interacts with the Arabidopsis SL signaling components. (A) Rice D14 efficiently bound Arabidopsis MAX2 in the presence of rac -GR24. Pull-down assay using recombinant His 6 -MAX2 and GST–D14 or GST–AtD14 in the absence or presence of rac -GR24. GST-fused proteins were detected by anti-GST antibody and the PVDF membrane was stained with MemStain to show equal loading. (B) Rice D14 efficiently bound Arabidopsis SMXL6 in the presence of rac -GR24. Pull-down assay using recombinant Flag-SMXL6 and GST–D14 or GST–AtD14 in the absence or presence of rac -GR24. GST-fused proteins were detected by anti-GST antibody and the PVDF membrane was stained with MemStain to show equal loading.

Techniques Used: Pull Down Assay, Recombinant, Staining

23) Product Images from "Cell surface localization of importin α1/KPNA2 affects cancer cell proliferation by regulating FGF1 signalling"

Article Title: Cell surface localization of importin α1/KPNA2 affects cancer cell proliferation by regulating FGF1 signalling

Journal: Scientific Reports

doi: 10.1038/srep21410

Importin α1 on the cell surface is functional. ( a ) Either GST-GFP or GST-NLS-GFP (50 pmol each) was incubated with 50 pmol of 3 × Flag-tagged importin α1 immobilized on glutathione beads. The bound proteins were analysed using immunoblotting with an anti-importin α1 or an anti-GST antibody, respectively. ( b ) Live HCT116 cells were incubated with GST-GFP or GST-NLS-GFP (1 μg/ml) for 1 h on ice, and we then observed fluorescence after fixation. Representative cells are shown. Scale bars: 10 μm. ( c ) Live HCT116 cells were treated with GST-GFP or GST-NLS-GFP (1 μg/ml) for 1 h on ice, and subjected to a flow cytometric analysis. ( d ) HCT116 cells transfected with control or importin α1 siRNAs, followed by treatment with GST-NLS-GFP (100 ng/ml) for 1 h on ice, and subjected to a flow cytometric analysis. ( e ) HCT116 cells were transfected with the empty vector (mock) or Flag-tagged importin α1 expression vector, followed by treatment with GST-GFP or GST-NLS-GFP (100 ng/ml) for 1 h on ice, and subjected to a flow cytometric analysis.
Figure Legend Snippet: Importin α1 on the cell surface is functional. ( a ) Either GST-GFP or GST-NLS-GFP (50 pmol each) was incubated with 50 pmol of 3 × Flag-tagged importin α1 immobilized on glutathione beads. The bound proteins were analysed using immunoblotting with an anti-importin α1 or an anti-GST antibody, respectively. ( b ) Live HCT116 cells were incubated with GST-GFP or GST-NLS-GFP (1 μg/ml) for 1 h on ice, and we then observed fluorescence after fixation. Representative cells are shown. Scale bars: 10 μm. ( c ) Live HCT116 cells were treated with GST-GFP or GST-NLS-GFP (1 μg/ml) for 1 h on ice, and subjected to a flow cytometric analysis. ( d ) HCT116 cells transfected with control or importin α1 siRNAs, followed by treatment with GST-NLS-GFP (100 ng/ml) for 1 h on ice, and subjected to a flow cytometric analysis. ( e ) HCT116 cells were transfected with the empty vector (mock) or Flag-tagged importin α1 expression vector, followed by treatment with GST-GFP or GST-NLS-GFP (100 ng/ml) for 1 h on ice, and subjected to a flow cytometric analysis.

Techniques Used: Functional Assay, Incubation, Fluorescence, Flow Cytometry, Transfection, Plasmid Preparation, Expressing

Importin α1 is localized at the cell surface of several cancer cell lines. ( a ) Total cell lysates of each cell line were subjected to an immunoblot analysis. GAPDH was used as a control. ( b ) Expression of importin α1 on the cell surface of several cancer cell lines, normal cell lines, and primary cells. Gray area, isotype control antibody (Ab). ( c ) Cell surface proteins on HCT116 cells, HepG2 cells, and AGS cells were biotinylated using NHS-biotin. Extracts of each fraction were pulled-down with neutravidin, and subjected to immunoblot analysis. Transferrin receptor (TfR) was used as a cell surface marker. Both GAPDH and Lamin A/C were used as a non-cell surface marker. ( d ) For conventional immunostaining, HCT116 cells were fixed, and subsequently permeabilized with 0.1% Triton X-100 for 5 min. For immunostaining under non-permeabilized conditions, living HCT116 cells were pretreated with extracellular tracer CtxB for 1 h on ice, fixed, and stained for importin α1. Representative cells are shown. Merged images were generated from Alexa488 staining (importin α1) and Alexa647 staining (CtxB). Co-localization is indicated in yellow. Scale bars: 10 μm. ( e ) Living HCT116 cells transfected with the empty vector (mock; blue line) or Flag-tagged importin α1 expression vector (red line) were stained with anti-Flag antibody, and subjected to flow cytometric analysis. ( f ) Living HCT116 cells transfected with the empty vector (mock; green line) or Flag-tagged importin α1 expression vector (red line) were stained with anti-importin α1 antibody, and subjected to flow cytometric analysis.
Figure Legend Snippet: Importin α1 is localized at the cell surface of several cancer cell lines. ( a ) Total cell lysates of each cell line were subjected to an immunoblot analysis. GAPDH was used as a control. ( b ) Expression of importin α1 on the cell surface of several cancer cell lines, normal cell lines, and primary cells. Gray area, isotype control antibody (Ab). ( c ) Cell surface proteins on HCT116 cells, HepG2 cells, and AGS cells were biotinylated using NHS-biotin. Extracts of each fraction were pulled-down with neutravidin, and subjected to immunoblot analysis. Transferrin receptor (TfR) was used as a cell surface marker. Both GAPDH and Lamin A/C were used as a non-cell surface marker. ( d ) For conventional immunostaining, HCT116 cells were fixed, and subsequently permeabilized with 0.1% Triton X-100 for 5 min. For immunostaining under non-permeabilized conditions, living HCT116 cells were pretreated with extracellular tracer CtxB for 1 h on ice, fixed, and stained for importin α1. Representative cells are shown. Merged images were generated from Alexa488 staining (importin α1) and Alexa647 staining (CtxB). Co-localization is indicated in yellow. Scale bars: 10 μm. ( e ) Living HCT116 cells transfected with the empty vector (mock; blue line) or Flag-tagged importin α1 expression vector (red line) were stained with anti-Flag antibody, and subjected to flow cytometric analysis. ( f ) Living HCT116 cells transfected with the empty vector (mock; green line) or Flag-tagged importin α1 expression vector (red line) were stained with anti-importin α1 antibody, and subjected to flow cytometric analysis.

Techniques Used: Expressing, Marker, Immunostaining, Staining, Generated, Transfection, Plasmid Preparation, Flow Cytometry

Importin α1 is released into the extracellular milieu by cell surface importin α1-positive cancer cells. ( a ) Subconfluent cancer cells were incubated with fresh medium. After 48 h, supernatants (upper panel) and lysates (lower panel) of cultured cancer cell lines were collected. Immunoblot analysis were performed using antibodies to importin α1 or Lamin A/C (as control). ( b ) Free importin α1 levels in the conditioned medium of cancer cell lines (HepG2 and AGS) were determined using ELISA. Data are means ± SD from three independent experiments. * P
Figure Legend Snippet: Importin α1 is released into the extracellular milieu by cell surface importin α1-positive cancer cells. ( a ) Subconfluent cancer cells were incubated with fresh medium. After 48 h, supernatants (upper panel) and lysates (lower panel) of cultured cancer cell lines were collected. Immunoblot analysis were performed using antibodies to importin α1 or Lamin A/C (as control). ( b ) Free importin α1 levels in the conditioned medium of cancer cell lines (HepG2 and AGS) were determined using ELISA. Data are means ± SD from three independent experiments. * P

Techniques Used: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Extracellular importin α1 binds to the cell surface via heparan sulfate. ( a ) HCT116 or AGS cells were incubated in the presence of PBS or recombinant Flag-tagged importin α1 (100 ng/ml) on ice for 1 h. Flag staining was visualized using confocal laser microscopy. Scale bars: 10 μm. ( b ) Living cancer cell lines were treated with PBS or recombinant importin α1 (100 ng/ml). These cells were stained with anti-importin α1 antibody (Ab), and subjected to flow cytometric analysis. Dashed line, isotype control Ab. ( c ) GST or GST-tagged importin α1 at concentrations of 0.5, 5, or 50 pmol were immobilized on heparin-beads. The bound proteins were analysed using immunoblotting with anti-GST Ab. GST-tagged-FGF1 and -FGF2 (50 pmol) were used as positive controls. ( d ) GST-tagged importin α1 mutants, GST-GFP or IBB domain-fused GST-GFP at concentrations of 50 pmol were immobilized on heparin beads. The bound proteins were analysed using immunoblotting with anti-GST Ab. Band intensities of each protein bound to heparin-sepharose were normalized with that of each input sample using the Image J program. ( e ) Supernatants of cultured cancer cell lines were collected and immobilized on heparin-conjugated beads. Immunoblot analysis was performed using Abs to importin α1. ( f ) Living HCT116 cells were treated with recombinant Flag-tagged importin α1 (100 ng/ml) in the presence or absence of heparin (10 U/ml). These cells were stained with anti-importin α1 Ab (Left panel) or anti-Flag antibody (Right panel), and subjected to flow cytometric analysis. Gray area, isotype control Ab. ( g ) Living HCT116 cells were pretreated with or without heparinase III (0.2 U/ml) for 6 h, and then treated with recombinant Flag-tagged importin α1 (100 ng/ml). These cells were stained with anti-importin α1 antibody, and subjected to flow cytometric analysis. Gray area, isotype control Ab.
Figure Legend Snippet: Extracellular importin α1 binds to the cell surface via heparan sulfate. ( a ) HCT116 or AGS cells were incubated in the presence of PBS or recombinant Flag-tagged importin α1 (100 ng/ml) on ice for 1 h. Flag staining was visualized using confocal laser microscopy. Scale bars: 10 μm. ( b ) Living cancer cell lines were treated with PBS or recombinant importin α1 (100 ng/ml). These cells were stained with anti-importin α1 antibody (Ab), and subjected to flow cytometric analysis. Dashed line, isotype control Ab. ( c ) GST or GST-tagged importin α1 at concentrations of 0.5, 5, or 50 pmol were immobilized on heparin-beads. The bound proteins were analysed using immunoblotting with anti-GST Ab. GST-tagged-FGF1 and -FGF2 (50 pmol) were used as positive controls. ( d ) GST-tagged importin α1 mutants, GST-GFP or IBB domain-fused GST-GFP at concentrations of 50 pmol were immobilized on heparin beads. The bound proteins were analysed using immunoblotting with anti-GST Ab. Band intensities of each protein bound to heparin-sepharose were normalized with that of each input sample using the Image J program. ( e ) Supernatants of cultured cancer cell lines were collected and immobilized on heparin-conjugated beads. Immunoblot analysis was performed using Abs to importin α1. ( f ) Living HCT116 cells were treated with recombinant Flag-tagged importin α1 (100 ng/ml) in the presence or absence of heparin (10 U/ml). These cells were stained with anti-importin α1 Ab (Left panel) or anti-Flag antibody (Right panel), and subjected to flow cytometric analysis. Gray area, isotype control Ab. ( g ) Living HCT116 cells were pretreated with or without heparinase III (0.2 U/ml) for 6 h, and then treated with recombinant Flag-tagged importin α1 (100 ng/ml). These cells were stained with anti-importin α1 antibody, and subjected to flow cytometric analysis. Gray area, isotype control Ab.

Techniques Used: Incubation, Recombinant, Staining, Microscopy, Flow Cytometry, Cell Culture

Extracellular importin α1 affects cell proliferation via interaction with FGF1 ( a ) GST, GST-FGF1, GST-FGF2, or GST-IGFBP5 (50 pmol) were incubated with 3 × Flag-importin α1 (50 pmol) immobilized on glutathione beads, and subjected to immunoblot analysis with the indicated antibodies. ( b ) Starved HCT116 cells were incubated in PBS or FGF1 (20 ng/ml) in the presence or absence of recombinant importin α1 at the indicated concentrations for 48 h. Cell proliferation was measured using Cell Counting Reagent. Data are means ± SD from three independent experiments. * P
Figure Legend Snippet: Extracellular importin α1 affects cell proliferation via interaction with FGF1 ( a ) GST, GST-FGF1, GST-FGF2, or GST-IGFBP5 (50 pmol) were incubated with 3 × Flag-importin α1 (50 pmol) immobilized on glutathione beads, and subjected to immunoblot analysis with the indicated antibodies. ( b ) Starved HCT116 cells were incubated in PBS or FGF1 (20 ng/ml) in the presence or absence of recombinant importin α1 at the indicated concentrations for 48 h. Cell proliferation was measured using Cell Counting Reagent. Data are means ± SD from three independent experiments. * P

Techniques Used: Incubation, Recombinant, Cell Counting

24) Product Images from "Role of the Salmonella Pathogenicity Island 1 (SPI-1) Protein InvB in Type III Secretion of SopE and SopE2, Two Salmonella Effector Proteins Encoded Outside of SPI-1"

Article Title: Role of the Salmonella Pathogenicity Island 1 (SPI-1) Protein InvB in Type III Secretion of SopE and SopE2, Two Salmonella Effector Proteins Encoded Outside of SPI-1

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.23.6950-6967.2003

(A) Western blot analysis of a GST-InvB affinity purification assay. GST-InvB (pM672) was expressed in wild-type Salmonella serovar Typhimurium SL1344 (see Materials and Methods). The bacterial lysate (25 ml) was incubated with 200 μl of GSH-Sepharose beads. Samples from the purification steps were analyzed by Western blotting by using anti-SopE (α-SopE), anti-SipA (α-SipA), and anti-SipC (α-SipC) antisera. Lane a, 100 μl of a whole culture before harvesting of the cells (wc); lane b, proteins recovered from 250 μl of culture supernatant after pelleting of the cells (sup); lane c, 25 μl of a bacterial pellet resuspended in 25 ml of buffer B; lane d, 50 μl of pelleted cell debris resuspended in 25 ml of buffer B after lysis with a French pressure cell (FP pe); lane e, 50 μl of cleared French pressure cell lysate (total volume, 25 ml) (FP sup); lane f, 50 μl of cleared cell lysate after binding of GST-InvB and its associated proteins to 200 μl of GSH-Sepharose beads (GSH sup); lanes g, 100 μl of washing solution after the first, fourth, and seventh washes of the GSH-Sepharose beads with buffer B (washing 1, 4, and 7, respectively); lane h, 10 μl of GSH-Sepharose beads. (B) Western blot analysis of a coimmunoprecipitation experiment performed with the sopE M45 -expressing strain M608 and an anti-M45 antibody (see Materials and Methods). The bacterial lysate (5 ml) was incubated with 10 μl of monoclonal mouse anti-M45 antibody and 10 μl of protein A-Sepharose beads. Samples from the precipitation procedure were analyzed by Western blotting by using polyclonal rabbit anti-InvB (rabbit α-InvB) and anti-SopE (rabbit α-SopE) antisera. Lane a, 100 μl of a whole culture before harvesting of the cells (wc); lane b, 100 μl of bacterial pellet resuspended in 20 ml of buffer B (pe); lane c, 100 μl of pelleted cell debris resuspended in 20 ml of buffer B after lysis with a French pressure cell (FP pe); lane d, 100 μl of cleared French pressure cell lysate (FP sup); lane e, 100 μl of cleared cell lysate after incubation with anti-M45 antibody and removal of nonspecific aggregates by centrifugation (α-m45 sup); lane f, 100 μl of pelleted nonspecific aggregates resuspended in buffer B (α-m45 pe); lane g, 100 μl of cleared cell lysate after incubation with 10 μl of protein A-Sepharose beads (prot. A sup); lane h, 100 μl of washing solution after the fourth wash of the protein A-Sepharose beads with buffer B (wash 4); lane i, 10 μl of protein A-Sepharose beads.
Figure Legend Snippet: (A) Western blot analysis of a GST-InvB affinity purification assay. GST-InvB (pM672) was expressed in wild-type Salmonella serovar Typhimurium SL1344 (see Materials and Methods). The bacterial lysate (25 ml) was incubated with 200 μl of GSH-Sepharose beads. Samples from the purification steps were analyzed by Western blotting by using anti-SopE (α-SopE), anti-SipA (α-SipA), and anti-SipC (α-SipC) antisera. Lane a, 100 μl of a whole culture before harvesting of the cells (wc); lane b, proteins recovered from 250 μl of culture supernatant after pelleting of the cells (sup); lane c, 25 μl of a bacterial pellet resuspended in 25 ml of buffer B; lane d, 50 μl of pelleted cell debris resuspended in 25 ml of buffer B after lysis with a French pressure cell (FP pe); lane e, 50 μl of cleared French pressure cell lysate (total volume, 25 ml) (FP sup); lane f, 50 μl of cleared cell lysate after binding of GST-InvB and its associated proteins to 200 μl of GSH-Sepharose beads (GSH sup); lanes g, 100 μl of washing solution after the first, fourth, and seventh washes of the GSH-Sepharose beads with buffer B (washing 1, 4, and 7, respectively); lane h, 10 μl of GSH-Sepharose beads. (B) Western blot analysis of a coimmunoprecipitation experiment performed with the sopE M45 -expressing strain M608 and an anti-M45 antibody (see Materials and Methods). The bacterial lysate (5 ml) was incubated with 10 μl of monoclonal mouse anti-M45 antibody and 10 μl of protein A-Sepharose beads. Samples from the precipitation procedure were analyzed by Western blotting by using polyclonal rabbit anti-InvB (rabbit α-InvB) and anti-SopE (rabbit α-SopE) antisera. Lane a, 100 μl of a whole culture before harvesting of the cells (wc); lane b, 100 μl of bacterial pellet resuspended in 20 ml of buffer B (pe); lane c, 100 μl of pelleted cell debris resuspended in 20 ml of buffer B after lysis with a French pressure cell (FP pe); lane d, 100 μl of cleared French pressure cell lysate (FP sup); lane e, 100 μl of cleared cell lysate after incubation with anti-M45 antibody and removal of nonspecific aggregates by centrifugation (α-m45 sup); lane f, 100 μl of pelleted nonspecific aggregates resuspended in buffer B (α-m45 pe); lane g, 100 μl of cleared cell lysate after incubation with 10 μl of protein A-Sepharose beads (prot. A sup); lane h, 100 μl of washing solution after the fourth wash of the protein A-Sepharose beads with buffer B (wash 4); lane i, 10 μl of protein A-Sepharose beads.

Techniques Used: Western Blot, Affinity Purification, Incubation, Purification, Lysis, Binding Assay, Expressing, Centrifugation

25) Product Images from "Lamin B Receptor Recognizes Specific Modifications of Histone H4 in Heterochromatin Formation *"

Article Title: Lamin B Receptor Recognizes Specific Modifications of Histone H4 in Heterochromatin Formation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.397950

LBR induces chromatin compaction. A , chromatin compaction was induced by LBR. Chromatin was reconstituted with 1.8-kbp linear dsDNA and core histones by salt dialysis. The chromatin was incubated with GST-LBR fragments, bound to mica, and then observed
Figure Legend Snippet: LBR induces chromatin compaction. A , chromatin compaction was induced by LBR. Chromatin was reconstituted with 1.8-kbp linear dsDNA and core histones by salt dialysis. The chromatin was incubated with GST-LBR fragments, bound to mica, and then observed

Techniques Used: Incubation

LBR specifically bind to H4K20me2. A , histone peptide array. Celluspot was incubated with 100 n m of GST-NP WT and probed with anti-GST antibody. The spots were detected by chemiluminescence. Detected spots related to H4K20 modifications are shown (see
Figure Legend Snippet: LBR specifically bind to H4K20me2. A , histone peptide array. Celluspot was incubated with 100 n m of GST-NP WT and probed with anti-GST antibody. The spots were detected by chemiluminescence. Detected spots related to H4K20 modifications are shown (see

Techniques Used: Peptide Microarray, Incubation

26) Product Images from "Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *"

Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.062364

Identification of Key Residues in Sortilin for Pro-domain Binding
Figure Legend Snippet: Identification of Key Residues in Sortilin for Pro-domain Binding

Techniques Used: Binding Assay

Mutational binding analysis of HisS-pro-BDNF to short sortilin fragments. A , HisS-BDNFpro binding analysis to peptides with the wild-type sortilin sequences 163 RIFRSSDF 170 and 170 FAKNFVQTD 178 listed to the left on each membrane. Binding to mutant peptides
Figure Legend Snippet: Mutational binding analysis of HisS-pro-BDNF to short sortilin fragments. A , HisS-BDNFpro binding analysis to peptides with the wild-type sortilin sequences 163 RIFRSSDF 170 and 170 FAKNFVQTD 178 listed to the left on each membrane. Binding to mutant peptides

Techniques Used: Binding Assay, Mutagenesis

Sortilin-derived peptide specifically competes binding of pro-NT to sortilin. A , recombinant sortilin was purified from 293 cells (indicated to the right by silver-stained SDS-PAGE analysis), and used for SPR studies to immobilized HisS-BDNFpro. The signal
Figure Legend Snippet: Sortilin-derived peptide specifically competes binding of pro-NT to sortilin. A , recombinant sortilin was purified from 293 cells (indicated to the right by silver-stained SDS-PAGE analysis), and used for SPR studies to immobilized HisS-BDNFpro. The signal

Techniques Used: Derivative Assay, Binding Assay, Recombinant, Purification, Staining, SDS Page, SPR Assay

Differential binding of pro-BDNF and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 n m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor
Figure Legend Snippet: Differential binding of pro-BDNF and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 n m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor

Techniques Used: Binding Assay, SPR Assay

Substitution analysis of the linear sortilin sequence. A , a representative substitutional binding analysis of HisS-BDNFpro to peptides with the wild-type sortilin sequence 163 RIFRSSDFAKNFVQTD 178 listed to the left on the membrane and detection using the
Figure Legend Snippet: Substitution analysis of the linear sortilin sequence. A , a representative substitutional binding analysis of HisS-BDNFpro to peptides with the wild-type sortilin sequence 163 RIFRSSDFAKNFVQTD 178 listed to the left on the membrane and detection using the

Techniques Used: Sequencing, Binding Assay

Identification of a linear pro-NT binding site within sortilin. A and B , sortilin represented as 273 overlapping peptides generated by SPOT synthesis on a cellulose membrane with peptides 64–69 binding to HisS-BDNFpro, as detected by either HRP-S-protein
Figure Legend Snippet: Identification of a linear pro-NT binding site within sortilin. A and B , sortilin represented as 273 overlapping peptides generated by SPOT synthesis on a cellulose membrane with peptides 64–69 binding to HisS-BDNFpro, as detected by either HRP-S-protein

Techniques Used: Binding Assay, Generated

GST-NGFpro binding to sortilin-WT and sortilin-4A. SPR analysis showing concentration series of GST-NGFpro (10, 20, 30, 40, and 50 n m ) tested for binding to immobilized extracellular domains of sortilin-4A ( A ) and sortilin-WT ( B ), demonstrating a strong
Figure Legend Snippet: GST-NGFpro binding to sortilin-WT and sortilin-4A. SPR analysis showing concentration series of GST-NGFpro (10, 20, 30, 40, and 50 n m ) tested for binding to immobilized extracellular domains of sortilin-4A ( A ) and sortilin-WT ( B ), demonstrating a strong

Techniques Used: Binding Assay, SPR Assay, Concentration Assay

Selective competition of ligands by sortilin-derived peptide antagonist. SPR binding analysis of 50 n m unprocessed pro-BDNF ( A ), 50 n m unprocessed pro-NGF ( B ), and 90 n m RAP ( C ) to immobilized sortilin in the absence and presence of the sort166–181
Figure Legend Snippet: Selective competition of ligands by sortilin-derived peptide antagonist. SPR binding analysis of 50 n m unprocessed pro-BDNF ( A ), 50 n m unprocessed pro-NGF ( B ), and 90 n m RAP ( C ) to immobilized sortilin in the absence and presence of the sort166–181

Techniques Used: Derivative Assay, SPR Assay, Binding Assay

Ligand Binding to Sortilin-4A
Figure Legend Snippet: Ligand Binding to Sortilin-4A

Techniques Used: Ligand Binding Assay

A sortilin-derived peptide blocks pro-NGF-induced cell death. A , RN22 schwannoma cells were incubated in the presence of 10 n m pro-NGF and increasing concentrations (0.2, 2.0, and 200 μ m ) of the sort166–181 peptide. The amount of pro-NGF-induced
Figure Legend Snippet: A sortilin-derived peptide blocks pro-NGF-induced cell death. A , RN22 schwannoma cells were incubated in the presence of 10 n m pro-NGF and increasing concentrations (0.2, 2.0, and 200 μ m ) of the sort166–181 peptide. The amount of pro-NGF-induced

Techniques Used: Derivative Assay, Incubation

Mutation of the linear binding site specifically impairs binding of both the NGF and the BDNF pro-domains. SPR analysis showing reduced binding of equal amounts (analyte concentration: 200 n m ) of the soluble extracellular domains of sortilin-4A compared
Figure Legend Snippet: Mutation of the linear binding site specifically impairs binding of both the NGF and the BDNF pro-domains. SPR analysis showing reduced binding of equal amounts (analyte concentration: 200 n m ) of the soluble extracellular domains of sortilin-4A compared

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

Decreased binding of pro-NT to sortilin-4A within cells. A , immunostaining of HEK 293 cells transfected with constructs for full-length ( fl )-sortilin-WT or sortilin-4A with an antibody against sortilin. Protein expression levels tested by Western blot
Figure Legend Snippet: Decreased binding of pro-NT to sortilin-4A within cells. A , immunostaining of HEK 293 cells transfected with constructs for full-length ( fl )-sortilin-WT or sortilin-4A with an antibody against sortilin. Protein expression levels tested by Western blot

Techniques Used: Binding Assay, Immunostaining, Transfection, Construct, Expressing, Western Blot

27) Product Images from "Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs) *"

Article Title: Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs) *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.O115.051839

Quantitative comparison of the binding affinity among seven different UBDs to ubiquitin and seven types of ubiquitin chains. A , flowchart for evaluating the binding affinity of recombinant UBDs to ubiquitin and ubiquitin chains with Western blot analysis and SILAC-AQUA. B , quantification of purified seven types of recombinant GST-UBDs on 10% SDS-polyacrylamide gels. A20_mut with the replacement of C35A and C38A was saved as the negative control for enrichment analysis. Same copy numbers of GST-UBDs were loaded on the gel and displayed with Coomassie Blue staining. C and D , comparison of the ubiquitin binding affinity of different UBDs. Equimolar UBDs were used to enrich ubiquitinated proteins from the same amount of yeast total cell lysates. The enriched ubiquitinated proteins were resolved by SDS-PAGE and probed with an anti-Myc antibody. The relative amount of ubiquitinated proteins was calculated based on intensity of the Western blot signal. E , quantitative comparison of the binding affinity of seven types of UBDs to seven types of ubiquitin chains. The amount of seven ubiquitin chains was measured by SILAC-AQUA. The same amount of heavy isotope-labeled ubiquitin chains purified by Ni-NTA under denaturing conditions was saved as the internal standard added to each reaction. The result was shown as relative amount compared with that from the enrichment of ubiquitinated proteins by Ni-NTA under denaturing conditions. Data are represented as mean and S.E.
Figure Legend Snippet: Quantitative comparison of the binding affinity among seven different UBDs to ubiquitin and seven types of ubiquitin chains. A , flowchart for evaluating the binding affinity of recombinant UBDs to ubiquitin and ubiquitin chains with Western blot analysis and SILAC-AQUA. B , quantification of purified seven types of recombinant GST-UBDs on 10% SDS-polyacrylamide gels. A20_mut with the replacement of C35A and C38A was saved as the negative control for enrichment analysis. Same copy numbers of GST-UBDs were loaded on the gel and displayed with Coomassie Blue staining. C and D , comparison of the ubiquitin binding affinity of different UBDs. Equimolar UBDs were used to enrich ubiquitinated proteins from the same amount of yeast total cell lysates. The enriched ubiquitinated proteins were resolved by SDS-PAGE and probed with an anti-Myc antibody. The relative amount of ubiquitinated proteins was calculated based on intensity of the Western blot signal. E , quantitative comparison of the binding affinity of seven types of UBDs to seven types of ubiquitin chains. The amount of seven ubiquitin chains was measured by SILAC-AQUA. The same amount of heavy isotope-labeled ubiquitin chains purified by Ni-NTA under denaturing conditions was saved as the internal standard added to each reaction. The result was shown as relative amount compared with that from the enrichment of ubiquitinated proteins by Ni-NTA under denaturing conditions. Data are represented as mean and S.E.

Techniques Used: Binding Assay, Recombinant, Western Blot, Purification, Negative Control, Staining, SDS Page, Labeling

28) Product Images from "The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases"

Article Title: The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases

Journal: PLoS ONE

doi: 10.1371/journal.pone.0161672

PDRG1 interacts with methionine adenosyltransferase α1. (A) Growth of yeast cotransfectants harboring pGBKT7-MATα1 (bait) and pACT2 plasmids (prey) including ORFs of MATα1, PDRG1, clone M2, clone M6 or laminin (negative control) in low (-LW) and high (-AHLW) stringency SC media. Additional controls including the empty pGBK plasmid are shown on the right. (B) Representative anti-FLAG immunoprecipitation results from four independent experiments using total lysates of CHO cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). The size of the standards is indicated on the left side of the panel. (C) Representative anti-HA immunoprecipitation data from three independent experiments utilizing total lysates of HEK 293T cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). Western blots of the input fractions were developed using anti-FLAG and anti-HA, whereas immunoprecipitates were analyzed using anti-HA or anti-FLAG with mouse TrueBlot ULTRA, as required. The arrow indicates an unspecific band recognized by anti-FLAG slightly over the FLAG-MATα1 signal in HEK 293T samples. The size of the standards is indicated on the left side of the panel. (D) Pull-down confirmation of the interaction using glutathione Sepharose beads loaded with GST or GST-PDRG1 and incubated with recombinant MATα1 plus excess GST. Results shown correspond to a typical experiments out of the five carried out; input fractions of the recombinant proteins used (left) and pull-down results (right) are shown. The size of the standards is indicated on the left side of the panel.
Figure Legend Snippet: PDRG1 interacts with methionine adenosyltransferase α1. (A) Growth of yeast cotransfectants harboring pGBKT7-MATα1 (bait) and pACT2 plasmids (prey) including ORFs of MATα1, PDRG1, clone M2, clone M6 or laminin (negative control) in low (-LW) and high (-AHLW) stringency SC media. Additional controls including the empty pGBK plasmid are shown on the right. (B) Representative anti-FLAG immunoprecipitation results from four independent experiments using total lysates of CHO cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). The size of the standards is indicated on the left side of the panel. (C) Representative anti-HA immunoprecipitation data from three independent experiments utilizing total lysates of HEK 293T cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). Western blots of the input fractions were developed using anti-FLAG and anti-HA, whereas immunoprecipitates were analyzed using anti-HA or anti-FLAG with mouse TrueBlot ULTRA, as required. The arrow indicates an unspecific band recognized by anti-FLAG slightly over the FLAG-MATα1 signal in HEK 293T samples. The size of the standards is indicated on the left side of the panel. (D) Pull-down confirmation of the interaction using glutathione Sepharose beads loaded with GST or GST-PDRG1 and incubated with recombinant MATα1 plus excess GST. Results shown correspond to a typical experiments out of the five carried out; input fractions of the recombinant proteins used (left) and pull-down results (right) are shown. The size of the standards is indicated on the left side of the panel.

Techniques Used: Negative Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Incubation, Recombinant

Pull-down analysis of PDRG1 interaction with MATα2 and MAT II. (A) Representative western blots of pull-down experiments using glutathione Sepharose beads loaded with GST or GST-PDRG1 and recombinant MATα2, MATβ or the hetero-oligomer MAT II; anti-GST, anti-MATα2 and MATβ were used for detection. The size of the standards is indicated on the left side of the panels. (B) Quantification of the MATα2/GST-PDRG1 signal ratio (mean ± SEM) from five independent pull-down experiments. (C) Representative western blots of pull-down experiments carried out with the truncated PDRG1 forms and recombinant MATα2 using anti-GST and anti-MATα2. The size of the standards is indicated on the left side of the panels. (D) Quantification of the MATα2/GST-PDRG1 signal ratio (mean ± SEM) from five independent pull-down experiments. All the incubations with MAT subunits or MAT II were carried out in the presence of excess GST to avoid unspecific binding. (*p≤0.05 vs GST-PDRG1).
Figure Legend Snippet: Pull-down analysis of PDRG1 interaction with MATα2 and MAT II. (A) Representative western blots of pull-down experiments using glutathione Sepharose beads loaded with GST or GST-PDRG1 and recombinant MATα2, MATβ or the hetero-oligomer MAT II; anti-GST, anti-MATα2 and MATβ were used for detection. The size of the standards is indicated on the left side of the panels. (B) Quantification of the MATα2/GST-PDRG1 signal ratio (mean ± SEM) from five independent pull-down experiments. (C) Representative western blots of pull-down experiments carried out with the truncated PDRG1 forms and recombinant MATα2 using anti-GST and anti-MATα2. The size of the standards is indicated on the left side of the panels. (D) Quantification of the MATα2/GST-PDRG1 signal ratio (mean ± SEM) from five independent pull-down experiments. All the incubations with MAT subunits or MAT II were carried out in the presence of excess GST to avoid unspecific binding. (*p≤0.05 vs GST-PDRG1).

Techniques Used: Western Blot, Recombinant, Binding Assay

Structural model of rat PDRG1 and interaction of PDRG1 truncated forms with MATα1. (A) PDRG1 structural model comprising residues K27-Q106 obtained with PHYRE. (B) Schematic representa tion of PDRG1 and the truncated forms prepared; the modeled area (white box) and deleted sequences (crossed box) are indicated. (C) Representative western blots of pull-down experiments carried out with recombinant truncated PDRG1 forms and MATα1 using anti-GST and anti-MATα1. Incubations with MATα1 were carried out in the presence of excess GST to avoid unspecific binding. The size of the standards is indicated on the left side of the panels. (D) Quantification of the MATα1/GST-PDRG1 signal ratio (mean ± SEM) from seven independent pull-down experiments (*p≤0.05 vs GST-PDRG1).
Figure Legend Snippet: Structural model of rat PDRG1 and interaction of PDRG1 truncated forms with MATα1. (A) PDRG1 structural model comprising residues K27-Q106 obtained with PHYRE. (B) Schematic representa tion of PDRG1 and the truncated forms prepared; the modeled area (white box) and deleted sequences (crossed box) are indicated. (C) Representative western blots of pull-down experiments carried out with recombinant truncated PDRG1 forms and MATα1 using anti-GST and anti-MATα1. Incubations with MATα1 were carried out in the presence of excess GST to avoid unspecific binding. The size of the standards is indicated on the left side of the panels. (D) Quantification of the MATα1/GST-PDRG1 signal ratio (mean ± SEM) from seven independent pull-down experiments (*p≤0.05 vs GST-PDRG1).

Techniques Used: Western Blot, Recombinant, Binding Assay

Effects of PDRG1 in DNA methylation and MAT activity. (A) Global DNA methylation levels of CHO cells transiently transfected with pHA-PDRG1, pFLAG-MAT or both plasmids evaluated with the inverse radioactive assay and compared to mock transfected cells. Incorporation of methyl groups into DNA (mean ± SEM) of five independent experiments carried out in triplicate is shown. For graphical purposes, the data are expressed as percentage of the pFLAG control taken as 100% (23392.65 ± 1790.07 cpm). Statistical analysis was performed using GraphPad Prism and changes were considered significant when p≤0.05 (*vs. pFLAG; ** vs. pHA; ***vs.FLAG-MAT). (B) Purified recombinant MATα1 (0.7 μM) was incubated with 0–5.6 μM PDRG1 (black) and S-adenosylmethionine synthesis determined; the panel shows results (mean ± SEM) of a typical experiment out of five carried out in triplicate. Controls including MATα1 and histone IIA (red) were also performed (C) Results (mean ± SEM) of a typical activity assay out of three performed in triplicate using MATα2 (0.7 μM). (D) Effects of PDRG1 (mean ± SEM) on MAT II activity (0.7 μM) from a typical experiment out of three carried out in triplicate. (E) Typical profile of a Biogel A purification of the MATα1/GST-PDRG1 complex followed by MAT activity. Elution of the standards is indicated with sticks that correspond to: Blue dextran (40 ml); ferritin (48 ml); aldolase (69 ml); conalbumin (81 ml); ovalbumin (84 ml); and ATP (105 ml). The upper part of the panel shows a stained SDS-PAGE gel of the relevant fractions as indicated on the top; the molecular size of the markers shown in the last lane (right) is indicated next to the corresponding stained band. (F) Comparison of the MAT activity shown by the MATα1/GST-PDRG1 (MATα1-HE; top) and MATα2/GST-PDRG1 complexes (MATα2-HE; bottom) vs. MATα1 or MATα2 homo-oligomers as correspond. The results shown are mean ± SEM of three independent experiments; *p
Figure Legend Snippet: Effects of PDRG1 in DNA methylation and MAT activity. (A) Global DNA methylation levels of CHO cells transiently transfected with pHA-PDRG1, pFLAG-MAT or both plasmids evaluated with the inverse radioactive assay and compared to mock transfected cells. Incorporation of methyl groups into DNA (mean ± SEM) of five independent experiments carried out in triplicate is shown. For graphical purposes, the data are expressed as percentage of the pFLAG control taken as 100% (23392.65 ± 1790.07 cpm). Statistical analysis was performed using GraphPad Prism and changes were considered significant when p≤0.05 (*vs. pFLAG; ** vs. pHA; ***vs.FLAG-MAT). (B) Purified recombinant MATα1 (0.7 μM) was incubated with 0–5.6 μM PDRG1 (black) and S-adenosylmethionine synthesis determined; the panel shows results (mean ± SEM) of a typical experiment out of five carried out in triplicate. Controls including MATα1 and histone IIA (red) were also performed (C) Results (mean ± SEM) of a typical activity assay out of three performed in triplicate using MATα2 (0.7 μM). (D) Effects of PDRG1 (mean ± SEM) on MAT II activity (0.7 μM) from a typical experiment out of three carried out in triplicate. (E) Typical profile of a Biogel A purification of the MATα1/GST-PDRG1 complex followed by MAT activity. Elution of the standards is indicated with sticks that correspond to: Blue dextran (40 ml); ferritin (48 ml); aldolase (69 ml); conalbumin (81 ml); ovalbumin (84 ml); and ATP (105 ml). The upper part of the panel shows a stained SDS-PAGE gel of the relevant fractions as indicated on the top; the molecular size of the markers shown in the last lane (right) is indicated next to the corresponding stained band. (F) Comparison of the MAT activity shown by the MATα1/GST-PDRG1 (MATα1-HE; top) and MATα2/GST-PDRG1 complexes (MATα2-HE; bottom) vs. MATα1 or MATα2 homo-oligomers as correspond. The results shown are mean ± SEM of three independent experiments; *p

Techniques Used: DNA Methylation Assay, Activity Assay, Transfection, Radioactivity, Purification, Recombinant, Incubation, Staining, SDS Page

29) Product Images from "Cytoplasmic Localization of Wis1 MAPKK by Nuclear Export Signal Is Important for Nuclear Targeting of Spc1/Sty1 MAPK in Fission Yeast"

Article Title: Cytoplasmic Localization of Wis1 MAPKK by Nuclear Export Signal Is Important for Nuclear Targeting of Spc1/Sty1 MAPK in Fission Yeast

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.02-03-0043

Residues 201–300 of Wis1 MAPKK are required for binding and phosphorylation of Spc1 MAPK. (A) Wis1ΔN300 is an active protein kinase. Bacterially produced GST-ΔN300 and GST-DDΔN300 proteins were tested for their autophosphorylation activity in the presence of [γ- 32 P]ATP. The samples were subjected to SDS-PAGE, followed by autoradiography. (B) Wis1ΔN300 cannot phosphorylate Spc1 in vitro. Activities of bacterially produced GST, GST-ΔN200, GST-ΔN300, or GST-DDΔN300 proteins were examined using GST-Spc1 as substrate. Phosphorylation of GST-Spc1 was detected by immunoblotting with anti-phospho-p38 antibodies. (C) Wis1ΔN300 cannot bind Spc1. Bacterially produced GST or different GST-Wis1 proteins were immobilized onto glutathione-beads and incubated with cell lysates from a spc1:myc strain (CA839). After extensive washes, proteins bound to the beads were analyzed with anti-GST and anti-myc antibodies.
Figure Legend Snippet: Residues 201–300 of Wis1 MAPKK are required for binding and phosphorylation of Spc1 MAPK. (A) Wis1ΔN300 is an active protein kinase. Bacterially produced GST-ΔN300 and GST-DDΔN300 proteins were tested for their autophosphorylation activity in the presence of [γ- 32 P]ATP. The samples were subjected to SDS-PAGE, followed by autoradiography. (B) Wis1ΔN300 cannot phosphorylate Spc1 in vitro. Activities of bacterially produced GST, GST-ΔN200, GST-ΔN300, or GST-DDΔN300 proteins were examined using GST-Spc1 as substrate. Phosphorylation of GST-Spc1 was detected by immunoblotting with anti-phospho-p38 antibodies. (C) Wis1ΔN300 cannot bind Spc1. Bacterially produced GST or different GST-Wis1 proteins were immobilized onto glutathione-beads and incubated with cell lysates from a spc1:myc strain (CA839). After extensive washes, proteins bound to the beads were analyzed with anti-GST and anti-myc antibodies.

Techniques Used: Binding Assay, Produced, Activity Assay, SDS Page, Autoradiography, In Vitro, Incubation

30) Product Images from "An Extended Helical Conformation in Domain 3a of Munc18-1 Provides a Template for SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Complex Assembly *"

Article Title: An Extended Helical Conformation in Domain 3a of Munc18-1 Provides a Template for SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Complex Assembly *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.514273

Binding of Munc18-1 constructs to syntaxin 1 and VAMP2. A , Munc18-1 mutants L348R, M334R, P335A, and P335A/L348R retain binding to monomeric syntaxin 1A. Immobilized GST-Munc18-1 constructs were incubated with the indicated amounts of syntaxin 1 for 1
Figure Legend Snippet: Binding of Munc18-1 constructs to syntaxin 1 and VAMP2. A , Munc18-1 mutants L348R, M334R, P335A, and P335A/L348R retain binding to monomeric syntaxin 1A. Immobilized GST-Munc18-1 constructs were incubated with the indicated amounts of syntaxin 1 for 1

Techniques Used: Binding Assay, Construct, Incubation

31) Product Images from "Auxin-induced SCFTIR1-Aux/IAA interaction involves stable modification of the SCFTIR1 complex"

Article Title: Auxin-induced SCFTIR1-Aux/IAA interaction involves stable modification of the SCFTIR1 complex

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

doi: 10.1073/pnas.0402868101

Auxin does not induce mass-shifting modifications within domain II. ( a and c ) MALDI/TOF MS spectra of trypsinized GST-AXR2dII exposed to plant extract without ( a ) and with ( c ) added auxin (10 μM NAA) ( n = 5). The 1,108-Da peak corresponding to the major part of the domain II degron and the derivative peak at +15.99 Da are indicated. ( b ) MALDI/TOF MS spectrum of trypsinized GST-AXR2dIIPP-AA (both prolines replaced by alanines). The new 1,056-Da peak corresponding to the major part of the mutant domain II degron is marked. For comparison, the positions at 1,056 + 15.99 Da, 1,108 Da, and 1,124 Da are indicated ( n = 2). n , the number of independent replicates.
Figure Legend Snippet: Auxin does not induce mass-shifting modifications within domain II. ( a and c ) MALDI/TOF MS spectra of trypsinized GST-AXR2dII exposed to plant extract without ( a ) and with ( c ) added auxin (10 μM NAA) ( n = 5). The 1,108-Da peak corresponding to the major part of the domain II degron and the derivative peak at +15.99 Da are indicated. ( b ) MALDI/TOF MS spectrum of trypsinized GST-AXR2dIIPP-AA (both prolines replaced by alanines). The new 1,056-Da peak corresponding to the major part of the mutant domain II degron is marked. For comparison, the positions at 1,056 + 15.99 Da, 1,108 Da, and 1,124 Da are indicated ( n = 2). n , the number of independent replicates.

Techniques Used: Mass Spectrometry, Mutagenesis

32) Product Images from "Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity"

Article Title: Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0106547

Effect of mutations in the N-terminal region of the SBD on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the proteins indicated at the top of each panel. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. ( a ) Both [S1-(P21H/A26P/Q62A)] NT [S2] CT and [S1-6Mut] NT [S2] CT chimeras only partially regained binding to PHD3, compared to binding of wild type Siah2 SBD to PHD3. ( b ) Only the chimera with the P21H/A26P mutations regained partial binding with PHD3. In contrast, mutation of Q62A did not increase PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.
Figure Legend Snippet: Effect of mutations in the N-terminal region of the SBD on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the proteins indicated at the top of each panel. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. ( a ) Both [S1-(P21H/A26P/Q62A)] NT [S2] CT and [S1-6Mut] NT [S2] CT chimeras only partially regained binding to PHD3, compared to binding of wild type Siah2 SBD to PHD3. ( b ) Only the chimera with the P21H/A26P mutations regained partial binding with PHD3. In contrast, mutation of Q62A did not increase PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.

Techniques Used: Binding Assay, Transfection, Expressing, Western Blot, Mutagenesis

Interaction of wild type (WT) and chimeric Siah proteins with PHD3. ( a ) Diagrammatic representation of the WT Siah1 and Siah2 SBD, which comprises of 1–193 residues, and the chimeric forms of Siah1 and Siah2 SBD, SBD[S1] NT [S2] CT and SBD[S2] NT [S1] CT . Corresponding original residue numbers are given in parentheses. ( b ) HEK293T cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. 48 hours after transfection, the cells were lysed and cell lysates were subjected to FLAG immunoprecipitation (IP). Immunopercipiates and the aliquotes of lysates were then immunoblotted using indicated antibodies. Both the chimeric forms lost binding to PHD3 as compared to wild type.
Figure Legend Snippet: Interaction of wild type (WT) and chimeric Siah proteins with PHD3. ( a ) Diagrammatic representation of the WT Siah1 and Siah2 SBD, which comprises of 1–193 residues, and the chimeric forms of Siah1 and Siah2 SBD, SBD[S1] NT [S2] CT and SBD[S2] NT [S1] CT . Corresponding original residue numbers are given in parentheses. ( b ) HEK293T cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. 48 hours after transfection, the cells were lysed and cell lysates were subjected to FLAG immunoprecipitation (IP). Immunopercipiates and the aliquotes of lysates were then immunoblotted using indicated antibodies. Both the chimeric forms lost binding to PHD3 as compared to wild type.

Techniques Used: Transfection, Cell Culture, Expressing, Immunoprecipitation, Binding Assay

Effect of mutations in the C-terminal region of the SBD on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S2] NT [S1-Q121L] CT chimera regained binding equivalent to Siah2 SBD wild type. In contrast, [S2] NT [S1-T160A] CT showed only a small increase in PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.
Figure Legend Snippet: Effect of mutations in the C-terminal region of the SBD on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S2] NT [S1-Q121L] CT chimera regained binding equivalent to Siah2 SBD wild type. In contrast, [S2] NT [S1-T160A] CT showed only a small increase in PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.

Techniques Used: Binding Assay, Transfection, Expressing, Immunoprecipitation, Western Blot

Docking model of the N-terminal Siah2 SBD and PHD3. N-terminal region (1–100) of the modeled Siah2 SBD was docked with PHD3 using an automated Cluspro server. The complex was then presented using Pymol as ( a ) cartoon representation, and ( b ) space filling representation. ( c ) The details of the interactions were obtained by PDBsum. The number of H-bond lines between any two residues indicates the number of potential hydrogen bonds between them. For non-bonded contacts, the width of the striped line is proportional to the number of atomic contacts.
Figure Legend Snippet: Docking model of the N-terminal Siah2 SBD and PHD3. N-terminal region (1–100) of the modeled Siah2 SBD was docked with PHD3 using an automated Cluspro server. The complex was then presented using Pymol as ( a ) cartoon representation, and ( b ) space filling representation. ( c ) The details of the interactions were obtained by PDBsum. The number of H-bond lines between any two residues indicates the number of potential hydrogen bonds between them. For non-bonded contacts, the width of the striped line is proportional to the number of atomic contacts.

Techniques Used:

Siah1 exhibits weak binding compared to Siah2 with PHD3. HEK293T cells were transfected in 60-mm cell culture plates for 2 days with expression plasmids for the proteins indicated at the top of each panel. ( a ) Cell lysates were subjected to HA-IP and aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-FLAG antibody. Both the Full length and Siah1 SBD did not show binding to PHD3 ( b ) The lysates were subjected to reciprocal FLAG-IP. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. In the IP, FLAG-SBD overlaps with the IgG light chain. Compared to Siah2 SBD, only weak binding of Siah1 SBD to PHD3 was observed.
Figure Legend Snippet: Siah1 exhibits weak binding compared to Siah2 with PHD3. HEK293T cells were transfected in 60-mm cell culture plates for 2 days with expression plasmids for the proteins indicated at the top of each panel. ( a ) Cell lysates were subjected to HA-IP and aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-FLAG antibody. Both the Full length and Siah1 SBD did not show binding to PHD3 ( b ) The lysates were subjected to reciprocal FLAG-IP. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. In the IP, FLAG-SBD overlaps with the IgG light chain. Compared to Siah2 SBD, only weak binding of Siah1 SBD to PHD3 was observed.

Techniques Used: Binding Assay, Transfection, Cell Culture, Expressing, Western Blot

Effect of mutations in Siah1 and Siah2 SBD Chimeras on binding with PHD3. ( a ) Pairwise sequence alignment of Siah1 and Siah2 SBD was performed by EMBOSS Needle tool. The 26 amino acids that are unique in Siah1 and Siah2 SBD are highlighted in grey. Dissimilar amino acids are highlighted by ‘*’. Similar amino acids are highlighted by ‘:’ and identical amino acids are highlighted by ‘|’ (top panel). The 10 dissimilar amino acids between Siah1 and Siah2 SBD are shown in diagrammatic representation of the chimeric forms, SBD[S1] NT [S2] CT and SBD[S1] NT [S2] CT (bottom panel). The original residue numbers are labeled in the respective colors ( b ) HEK293T cells were transfected with the indicated expression plasmids, followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The N-terminal mutant chimera, [S1-(E17S/P57S/F98H)] NT [S2] CT did not regain binding to PHD3 and the C-terminal mutant chimera, [S2] NT [S1-(Q121L/T160A] CT regained complete binding to PHD3 equivalent to WT Siah2 SBD.
Figure Legend Snippet: Effect of mutations in Siah1 and Siah2 SBD Chimeras on binding with PHD3. ( a ) Pairwise sequence alignment of Siah1 and Siah2 SBD was performed by EMBOSS Needle tool. The 26 amino acids that are unique in Siah1 and Siah2 SBD are highlighted in grey. Dissimilar amino acids are highlighted by ‘*’. Similar amino acids are highlighted by ‘:’ and identical amino acids are highlighted by ‘|’ (top panel). The 10 dissimilar amino acids between Siah1 and Siah2 SBD are shown in diagrammatic representation of the chimeric forms, SBD[S1] NT [S2] CT and SBD[S1] NT [S2] CT (bottom panel). The original residue numbers are labeled in the respective colors ( b ) HEK293T cells were transfected with the indicated expression plasmids, followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The N-terminal mutant chimera, [S1-(E17S/P57S/F98H)] NT [S2] CT did not regain binding to PHD3 and the C-terminal mutant chimera, [S2] NT [S1-(Q121L/T160A] CT regained complete binding to PHD3 equivalent to WT Siah2 SBD.

Techniques Used: Binding Assay, Sequencing, Labeling, Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

Effect of additional mutations in the N-terminal region of the SBD on binding with PHD3. ( a ) The 10 similar amino acids in the N terminal region (1–00) of Siah1 and Siah2 SBD are highlighted in grey. Mutated residues among the similar amino acids are highlighted within the box. ( b ) HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S1-8Mut] NT [S2] CT mutant increased binding compared to [S1-6Mut] NT [S2] CT . ( c ) The amount of PHD3 that coimmunoprecipitated with chimeric and mutated Siah1 and Siah2 SBD was quantified using Gel-pro analyzer software. The binding of the chimeric and mutated SBD to PHD3 was expressed as percentage of the binding of WT Siah2 SBD to PHD3. The data are represented as mean±S.E.M from three independent experiments. Differences in measured variables were assessed with Student's t test. * denotes p
Figure Legend Snippet: Effect of additional mutations in the N-terminal region of the SBD on binding with PHD3. ( a ) The 10 similar amino acids in the N terminal region (1–00) of Siah1 and Siah2 SBD are highlighted in grey. Mutated residues among the similar amino acids are highlighted within the box. ( b ) HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S1-8Mut] NT [S2] CT mutant increased binding compared to [S1-6Mut] NT [S2] CT . ( c ) The amount of PHD3 that coimmunoprecipitated with chimeric and mutated Siah1 and Siah2 SBD was quantified using Gel-pro analyzer software. The binding of the chimeric and mutated SBD to PHD3 was expressed as percentage of the binding of WT Siah2 SBD to PHD3. The data are represented as mean±S.E.M from three independent experiments. Differences in measured variables were assessed with Student's t test. * denotes p

Techniques Used: Binding Assay, Transfection, Expressing, Western Blot, Mutagenesis, Software

Interaction of Siah2 with PHD3. ( a ) HEK293 cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. The cells were lysed, and the lysates were subjected to FLAG immunoprecipitation (IP), as described under “ Materials and Methods ”. Aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-HA antibody. Both full length Siah2 and Siah2 SBD bind to PHD3 to the same extent. In the IP, the presence of the faint band in the empty vector lane is due to non-specific binding of PHD3. The same membrane was reblotted with FLAG antibody to detect FLAG tagged Siah2 proteins. ( b ) GST-Siah2 SBD pulldown of HA-PHD3. Cell lysate of HEK293 cells transfected with HA-PHD3 was incubated with GST-Siah2 SBD immobilized on GSH agarose beads and the reaction was performed as described under “ Material and Methods ”. The empty expression vector alone was expressed as a GST control for non-specific binding of HA PHD3. After the incubation, the lysate was removed, the GSH-agarose beads were washed, and bound HA-PHD3 was analyzed by Western blotting using anti HA antibody. The pull down assay confirmed the interaction of Siah2 SBD with PHD3.
Figure Legend Snippet: Interaction of Siah2 with PHD3. ( a ) HEK293 cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. The cells were lysed, and the lysates were subjected to FLAG immunoprecipitation (IP), as described under “ Materials and Methods ”. Aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-HA antibody. Both full length Siah2 and Siah2 SBD bind to PHD3 to the same extent. In the IP, the presence of the faint band in the empty vector lane is due to non-specific binding of PHD3. The same membrane was reblotted with FLAG antibody to detect FLAG tagged Siah2 proteins. ( b ) GST-Siah2 SBD pulldown of HA-PHD3. Cell lysate of HEK293 cells transfected with HA-PHD3 was incubated with GST-Siah2 SBD immobilized on GSH agarose beads and the reaction was performed as described under “ Material and Methods ”. The empty expression vector alone was expressed as a GST control for non-specific binding of HA PHD3. After the incubation, the lysate was removed, the GSH-agarose beads were washed, and bound HA-PHD3 was analyzed by Western blotting using anti HA antibody. The pull down assay confirmed the interaction of Siah2 SBD with PHD3.

Techniques Used: Transfection, Cell Culture, Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, Binding Assay, Incubation, Pull Down Assay

33) Product Images from "The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation"

Article Title: The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1002480

The structure of the TPR domain of Rrp5. (A) Cartoon representation of the crystal structure. (B) Surface charge distribution of the TPR domain of Rrp5. (C) Surface representation of amino acid conservation calculated using the ConSurf server [ 35 ] and displayed in PyMOL (The PyMOL Molecular Graphics System, Version 1.0r1 Schrödinger). The program ConSurf uses the provided sequence ( S . cerevisiae Rrp5) to find the 150 closest homologs (lowest E-values) for generating a multiple sequence alignment. The organisms used for Rrp5-TPR can be found here: http://consurf.tau.ac.il/results/1463756501/query_final_homolougs.html . The conserved patches on the surface of the TPR motif that interact with Rok1 are marked in red. (D) Mutations in conserved charged areas of the TPR domain are deleterious in vivo. TPR2: E1509K/E1510K/E1512K and TPR7: K1686E/K1689E. (E) Mutations in conserved charged areas of the TPR domain weaken the Rok1•Rrp5 interaction in vitro. (F) Position of the TPR mutations in the 3-D reconstruction of the Rrp5•Rok1 complex. (G) Recombinant GST-Rok1 immobilized on glutathione (GSH) sepharose resin interacts with recombinant, purified Rrp5 FL , Rrp5_C7, and Rrp5_C6, but not Rrp5_C5. (H) Maltose binding protein (MBP) or glutathione S-transferase (GST) alone does not bind to the Rrp5 fragments tested here. I, input; W, wash; E, elution.
Figure Legend Snippet: The structure of the TPR domain of Rrp5. (A) Cartoon representation of the crystal structure. (B) Surface charge distribution of the TPR domain of Rrp5. (C) Surface representation of amino acid conservation calculated using the ConSurf server [ 35 ] and displayed in PyMOL (The PyMOL Molecular Graphics System, Version 1.0r1 Schrödinger). The program ConSurf uses the provided sequence ( S . cerevisiae Rrp5) to find the 150 closest homologs (lowest E-values) for generating a multiple sequence alignment. The organisms used for Rrp5-TPR can be found here: http://consurf.tau.ac.il/results/1463756501/query_final_homolougs.html . The conserved patches on the surface of the TPR motif that interact with Rok1 are marked in red. (D) Mutations in conserved charged areas of the TPR domain are deleterious in vivo. TPR2: E1509K/E1510K/E1512K and TPR7: K1686E/K1689E. (E) Mutations in conserved charged areas of the TPR domain weaken the Rok1•Rrp5 interaction in vitro. (F) Position of the TPR mutations in the 3-D reconstruction of the Rrp5•Rok1 complex. (G) Recombinant GST-Rok1 immobilized on glutathione (GSH) sepharose resin interacts with recombinant, purified Rrp5 FL , Rrp5_C7, and Rrp5_C6, but not Rrp5_C5. (H) Maltose binding protein (MBP) or glutathione S-transferase (GST) alone does not bind to the Rrp5 fragments tested here. I, input; W, wash; E, elution.

Techniques Used: Sequencing, In Vivo, In Vitro, Recombinant, Purification, Binding Assay

34) Product Images from "In vitro oxidative inactivation of human presequence protease (hPreP)"

Article Title: In vitro oxidative inactivation of human presequence protease (hPreP)

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2012.09.039

Effect of exposure to hydrogen peroxide on hPreP wild-type activity. After 4 h incubation with H 2 O 2 , the hydrogen peroxide was removed and hPreP activity assayed as the cleavage of four substrates. (A) Representative gel showing cleavage of C1 by hPreP, resulting in a change in migration on agarose gel due to the charge profile of the peptide. (B) Effect of oxidation on the rate of substrate V degradation by hPreP (average of three experiments). Time course analysis of (C) pF 1 β and (D) Aβ degradation by hPreP incubated in the absence or presence (0.5 or 5 mM) of H 2 O 2 . Shown are representative gels (corresponding to one of the three experiments) and an estimation of the degradation rate (in the first 10 min for pF 1 β and in the first 45 min for Aβ).
Figure Legend Snippet: Effect of exposure to hydrogen peroxide on hPreP wild-type activity. After 4 h incubation with H 2 O 2 , the hydrogen peroxide was removed and hPreP activity assayed as the cleavage of four substrates. (A) Representative gel showing cleavage of C1 by hPreP, resulting in a change in migration on agarose gel due to the charge profile of the peptide. (B) Effect of oxidation on the rate of substrate V degradation by hPreP (average of three experiments). Time course analysis of (C) pF 1 β and (D) Aβ degradation by hPreP incubated in the absence or presence (0.5 or 5 mM) of H 2 O 2 . Shown are representative gels (corresponding to one of the three experiments) and an estimation of the degradation rate (in the first 10 min for pF 1 β and in the first 45 min for Aβ).

Techniques Used: Activity Assay, Incubation, Migration, Agarose Gel Electrophoresis

35) Product Images from "NF-?B1 p105 Negatively Regulates TPL-2 MEK Kinase Activity"

Article Title: NF-?B1 p105 Negatively Regulates TPL-2 MEK Kinase Activity

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.14.4739-4752.2003

p105 blocks interaction between TPL-2 and MEK. (A) 3T3 fibroblasts were transfected with EV or TPL-2 vector or in combination with HA-p105 vector or EV. Cell lysates were Western blotted and probed sequentially with the indicated antibodies. (B) 293 cells were transfected with expression vectors encoding Myc-TPL-2 with or without wild-type or mutant version of HA-p105. GST-MEK1(K207A) protein, bound to GSH-Sepharose 4B beads, was used to affinity purify Myc-TPL-2 from cell lysates. Isolated protein and protein expression in lysates was assayed by Western blotting. (C) Lysates from 293 cells transfected with Myc-TPL-2 were incubated with the indicated concentrations of p105 DD protein prior to addition of GST-MEK1(K207A) affinity ligand. Pulldowns and lysates were Western blotted.
Figure Legend Snippet: p105 blocks interaction between TPL-2 and MEK. (A) 3T3 fibroblasts were transfected with EV or TPL-2 vector or in combination with HA-p105 vector or EV. Cell lysates were Western blotted and probed sequentially with the indicated antibodies. (B) 293 cells were transfected with expression vectors encoding Myc-TPL-2 with or without wild-type or mutant version of HA-p105. GST-MEK1(K207A) protein, bound to GSH-Sepharose 4B beads, was used to affinity purify Myc-TPL-2 from cell lysates. Isolated protein and protein expression in lysates was assayed by Western blotting. (C) Lysates from 293 cells transfected with Myc-TPL-2 were incubated with the indicated concentrations of p105 DD protein prior to addition of GST-MEK1(K207A) affinity ligand. Pulldowns and lysates were Western blotted.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Expressing, Mutagenesis, Isolation, Incubation

36) Product Images from "The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage"

Article Title: The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg459

Fig. 1. The 35 C-terminal amino acids of Stu2 mediate binding to Spc72. ( A ), the coiled coil domain was predicted from amino acids 697–758 and the low complexity region (LC) from amino acids 766–781. Abbreviations: –, +, ++ symbolize no, good or strong interaction in the two-hybrid system, respectively. ( B ) Stu2 1–855 failed to bind to N-Spc72 in vitro . Sf9 cell extracts with Stu2 or Stu2 1–855 (lanes 1 and 2) were incubated with E.coli -expressed purified GST (lanes 3 and 5) or GST–N-Spc72 (lanes 4 and 6) bound to glutathione–Sepharose beads. After washing, the bound proteins were eluted and analysed by immunoblotting.
Figure Legend Snippet: Fig. 1. The 35 C-terminal amino acids of Stu2 mediate binding to Spc72. ( A ), the coiled coil domain was predicted from amino acids 697–758 and the low complexity region (LC) from amino acids 766–781. Abbreviations: –, +, ++ symbolize no, good or strong interaction in the two-hybrid system, respectively. ( B ) Stu2 1–855 failed to bind to N-Spc72 in vitro . Sf9 cell extracts with Stu2 or Stu2 1–855 (lanes 1 and 2) were incubated with E.coli -expressed purified GST (lanes 3 and 5) or GST–N-Spc72 (lanes 4 and 6) bound to glutathione–Sepharose beads. After washing, the bound proteins were eluted and analysed by immunoblotting.

Techniques Used: Binding Assay, In Vitro, Incubation, Purification

Fig. 6. The Tub4 complex, Stu2 and Spc72 assemble into one complex and act in a cooperative manner. ( A ) Tub4 complex, Stu2 and Spc72 are part of common complexes. Lysates of logarithmically growing or α-factor arrested SPC97 and SPC97-3HA cells, both with STU2-3Myc , were subjected to anti-HA immunoprecipitation. Precipitated proteins were detected by immunoblotting with the indicated antibodies. ( B ) Tub4 complex (lanes 1–3 and 7–9) and Stu2-3HA (lanes 4–9), both from Sf9 cells, were incubated with recombinant and purified GST or GST–N-Spc72. After an initial incubation step, anti-HA antibodies were added followed by protein G Sepharose beads. The anti-HA precipitates were analysed by immunoblotting with the indicated antibodies. ( C ) Genetic interaction between SPC98 and SPC72 ΔStu2 . Serial dilutions of the indicated yeast cells were grown at the indicated temperatures on YPD plates for 3 days.
Figure Legend Snippet: Fig. 6. The Tub4 complex, Stu2 and Spc72 assemble into one complex and act in a cooperative manner. ( A ) Tub4 complex, Stu2 and Spc72 are part of common complexes. Lysates of logarithmically growing or α-factor arrested SPC97 and SPC97-3HA cells, both with STU2-3Myc , were subjected to anti-HA immunoprecipitation. Precipitated proteins were detected by immunoblotting with the indicated antibodies. ( B ) Tub4 complex (lanes 1–3 and 7–9) and Stu2-3HA (lanes 4–9), both from Sf9 cells, were incubated with recombinant and purified GST or GST–N-Spc72. After an initial incubation step, anti-HA antibodies were added followed by protein G Sepharose beads. The anti-HA precipitates were analysed by immunoblotting with the indicated antibodies. ( C ) Genetic interaction between SPC98 and SPC72 ΔStu2 . Serial dilutions of the indicated yeast cells were grown at the indicated temperatures on YPD plates for 3 days.

Techniques Used: Activated Clotting Time Assay, Immunoprecipitation, Incubation, Recombinant, Purification

37) Product Images from "Unique self-palmitoylation activity of the transport protein particle component Bet3: A mechanism required for protein stability"

Article Title: Unique self-palmitoylation activity of the transport protein particle component Bet3: A mechanism required for protein stability

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

doi: 10.1073/pnas.0603513103

Bet3 binds Pal-CoA at physiological concentrations. ( A ) His-Bet3-StrepII purified from E. coli was added to Pal-CoA agarose. Washed beads were eluted twice with 10 mM Pal-CoA and finally with SDS buffer. Comparable aliquots of input (IP), flow-through (FT), wash 1 (W1), wash 4 (W4), wash 9 (W9), and from eluates were subjected to SDS/PAGE and Coomassie staining. wt, wild type. ( B ) Bet3 purified from E. coli was incubated with Cibacron Blue Sepharose for 1 h. Beads were washed and incubated with increasing concentrations of Pal-CoA ( Upper ) or with CoA ( Lower ). Comparable aliquots of input (IP), flow-through (FT), and eluates were subjected to SDS/PAGE and Coomassie staining. ( C ) Bet3 C68S (CS) was incubated with Pal-CoA agarose ( Upper ) and Cibacron Blue Sepharose ( Lower ) and processed as described in A and B . ( D ) Soluble Cibacron Blue at the concentration indicated was added to the palmitoylation reaction with GST-Bet3 purified from E. coli . The resulting fluorogram is shown. ( E ) Bet3 from yeast (2 μM) was incubated with increasing concentrations of [ 3 H]Pal-CoA for 5 min at 30°C. ( Inset ) The resulting fluorogram was quantified by densitometry, and the rate of palmitoylation in arbitrary units was plotted against the [ 3 H]Pal-CoA concentration. The data points were fitted according to the Michaelis–Menten equation v = v max × [ S ]/ K m + [ S ], and the K m of Bet3 for Pal-CoA was calculated as 5.7 μM.
Figure Legend Snippet: Bet3 binds Pal-CoA at physiological concentrations. ( A ) His-Bet3-StrepII purified from E. coli was added to Pal-CoA agarose. Washed beads were eluted twice with 10 mM Pal-CoA and finally with SDS buffer. Comparable aliquots of input (IP), flow-through (FT), wash 1 (W1), wash 4 (W4), wash 9 (W9), and from eluates were subjected to SDS/PAGE and Coomassie staining. wt, wild type. ( B ) Bet3 purified from E. coli was incubated with Cibacron Blue Sepharose for 1 h. Beads were washed and incubated with increasing concentrations of Pal-CoA ( Upper ) or with CoA ( Lower ). Comparable aliquots of input (IP), flow-through (FT), and eluates were subjected to SDS/PAGE and Coomassie staining. ( C ) Bet3 C68S (CS) was incubated with Pal-CoA agarose ( Upper ) and Cibacron Blue Sepharose ( Lower ) and processed as described in A and B . ( D ) Soluble Cibacron Blue at the concentration indicated was added to the palmitoylation reaction with GST-Bet3 purified from E. coli . The resulting fluorogram is shown. ( E ) Bet3 from yeast (2 μM) was incubated with increasing concentrations of [ 3 H]Pal-CoA for 5 min at 30°C. ( Inset ) The resulting fluorogram was quantified by densitometry, and the rate of palmitoylation in arbitrary units was plotted against the [ 3 H]Pal-CoA concentration. The data points were fitted according to the Michaelis–Menten equation v = v max × [ S ]/ K m + [ S ], and the K m of Bet3 for Pal-CoA was calculated as 5.7 μM.

Techniques Used: Purification, Flow Cytometry, SDS Page, Staining, Incubation, Concentration Assay

38) Product Images from "Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway"

Article Title: Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway

Journal: Journal of Virology

doi: 10.1128/JVI.01684-19

SFTSV NSs inhibits the TRIM21-p62 interaction. (A and B) Mapping of TRIM21 for NSs binding. (A) HEK293T cells were transfected with NSs-3×Flag and the individual domain (RBCC [R, ring; B, B box; CC, coiled-coil] and PRY/SPRY) of TRIM21-V5, and WCEs were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. (B) HEK293T cells were transfected with NSs-GST and the individual domain (PRY/SPRY, PRY, or SPRY) of TRIM21-V5, and WCEs were pulled down by glutathione beads, followed by immunoblotting with the indicated antibody. (C) A model of the molecular action of NSs in TRIM21 function. Ub, ubiquitin. (D) HEK293T cells were transfected with increasing amount of NSs-3×Flag, TRIM21-V5, and p62-Myc, and WCEs were immunoprecipitated by anti-V5 antibody, followed by immunoblotting with the indicated antibody. (E) HeLa cells were transfected with NSs-3×Flag-GFP, TRIM21-V5, and p62-Myc. The cells were fixed and stained with primary antibodies (rabbit anti-V5 or mouse anti-Myc) and with secondary antibodies (Alexa Fluor 568-conjugated anti-rabbit IgG or Alexa Fluor 350-conjugated mouse IgG) for confocal microscopy. No nucleus staining was performed. (F) HeLa cells were transfected with NSs-3×Flag-GFP and p62-Myc. The cells were fixed and stained with primary antibody (mouse anti-Myc) and with secondary antibody (Alexa Fluor 568-conjugated anti-mouse IgG) for confocal microscopy. Hoechst staining was used for the nucleus. The microscope images represent those from three independent experiments. (G) HEK293T cells were transfected with NSs-V5 and Keap1-Flag, and WCEs were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting with the indicated antibody.
Figure Legend Snippet: SFTSV NSs inhibits the TRIM21-p62 interaction. (A and B) Mapping of TRIM21 for NSs binding. (A) HEK293T cells were transfected with NSs-3×Flag and the individual domain (RBCC [R, ring; B, B box; CC, coiled-coil] and PRY/SPRY) of TRIM21-V5, and WCEs were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. (B) HEK293T cells were transfected with NSs-GST and the individual domain (PRY/SPRY, PRY, or SPRY) of TRIM21-V5, and WCEs were pulled down by glutathione beads, followed by immunoblotting with the indicated antibody. (C) A model of the molecular action of NSs in TRIM21 function. Ub, ubiquitin. (D) HEK293T cells were transfected with increasing amount of NSs-3×Flag, TRIM21-V5, and p62-Myc, and WCEs were immunoprecipitated by anti-V5 antibody, followed by immunoblotting with the indicated antibody. (E) HeLa cells were transfected with NSs-3×Flag-GFP, TRIM21-V5, and p62-Myc. The cells were fixed and stained with primary antibodies (rabbit anti-V5 or mouse anti-Myc) and with secondary antibodies (Alexa Fluor 568-conjugated anti-rabbit IgG or Alexa Fluor 350-conjugated mouse IgG) for confocal microscopy. No nucleus staining was performed. (F) HeLa cells were transfected with NSs-3×Flag-GFP and p62-Myc. The cells were fixed and stained with primary antibody (mouse anti-Myc) and with secondary antibody (Alexa Fluor 568-conjugated anti-mouse IgG) for confocal microscopy. Hoechst staining was used for the nucleus. The microscope images represent those from three independent experiments. (G) HEK293T cells were transfected with NSs-V5 and Keap1-Flag, and WCEs were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting with the indicated antibody.

Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Staining, Confocal Microscopy, Microscopy

NSs mutants for interacting with three different host factors. (A to C) (Top) HEK293T cells were transfected with TRIM21-V5 and NSs-GST for four NSs (NSs-WT, NSs-A46, NSs-PA, and NSs-KR) (A), ABIN2-V5 and NSs-3×Flag for three NSs (NSs-WT, NSs-A46, and NSs-KR) (B), and TBK1-Flag with NSs-V5 for three NSs (NSs-WT, NSs-A46, and NSs-PA) (C). (Bottom) WCEs were pulled down by glutathione beads (A) or immunoprecipitated by anti-Flag antibody (B) or anti-V5 antibody (C), followed by immunoblotting with the indicated antibody.
Figure Legend Snippet: NSs mutants for interacting with three different host factors. (A to C) (Top) HEK293T cells were transfected with TRIM21-V5 and NSs-GST for four NSs (NSs-WT, NSs-A46, NSs-PA, and NSs-KR) (A), ABIN2-V5 and NSs-3×Flag for three NSs (NSs-WT, NSs-A46, and NSs-KR) (B), and TBK1-Flag with NSs-V5 for three NSs (NSs-WT, NSs-A46, and NSs-PA) (C). (Bottom) WCEs were pulled down by glutathione beads (A) or immunoprecipitated by anti-Flag antibody (B) or anti-V5 antibody (C), followed by immunoblotting with the indicated antibody.

Techniques Used: Transfection, Immunoprecipitation

NSs interacts with TRIM21. (A) HEK293T cells were transfected with NSs-GST and TRIM21-V5, and whole-cell extracts (WCEs) were pulled down by glutathione beads, followed by immunoblotting with the indicated antibody. (B) HEK293T cells were transfected with NSs-3×Flag and TRIM21-V5, and WCEs were applied to SiMPull analysis. (Left) Three representative images. (Right) Molecular numbers, in which the bar graphs indicate the average number of fluorophores per image. Error bars represent the SD of the mean across > 20 images. The results of three independent experiments are represented. (C) RAW 264.7 cells were infected with SFTSV-NSs-GFP, a recombinant virus expressing GFP-tagged NSs, for 24 h and subjected to immunoprecipitation (IP) with anti-GFP antibody to pull down the GFP-NSs complex, followed by immunoblotting with anti-TRIM21 antibody to detect endogenous TRIM21. (D) HeLa cells were transfected with NSs-3×Flag-GFP and TRIM21-V5. The cells were fixed and stained with primary mouse anti-V5 antibody and with secondary Alexa Fluor 568-conjugated anti-mouse IgG antibody for confocal microscopy. Hoechst staining was used for the nucleus. The microscope images represent those from three independent experiments.
Figure Legend Snippet: NSs interacts with TRIM21. (A) HEK293T cells were transfected with NSs-GST and TRIM21-V5, and whole-cell extracts (WCEs) were pulled down by glutathione beads, followed by immunoblotting with the indicated antibody. (B) HEK293T cells were transfected with NSs-3×Flag and TRIM21-V5, and WCEs were applied to SiMPull analysis. (Left) Three representative images. (Right) Molecular numbers, in which the bar graphs indicate the average number of fluorophores per image. Error bars represent the SD of the mean across > 20 images. The results of three independent experiments are represented. (C) RAW 264.7 cells were infected with SFTSV-NSs-GFP, a recombinant virus expressing GFP-tagged NSs, for 24 h and subjected to immunoprecipitation (IP) with anti-GFP antibody to pull down the GFP-NSs complex, followed by immunoblotting with anti-TRIM21 antibody to detect endogenous TRIM21. (D) HeLa cells were transfected with NSs-3×Flag-GFP and TRIM21-V5. The cells were fixed and stained with primary mouse anti-V5 antibody and with secondary Alexa Fluor 568-conjugated anti-mouse IgG antibody for confocal microscopy. Hoechst staining was used for the nucleus. The microscope images represent those from three independent experiments.

Techniques Used: Transfection, Infection, Recombinant, Expressing, Immunoprecipitation, Staining, Confocal Microscopy, Microscopy

TRIM21-binding-deficient NSs mutant. (A) Schematic diagram of the NSs-A46 mutant carrying alanine substitutions at K 226 KTDG 230 . (B) HEK293T cells were transfected with NSs-WT-3×Flag, NSs-A46-3×Flag, and TRIM21-V5, and WCEs were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. (C) HEK293T cells were transfected with pEBG-GST, GST-NSs-WT, and GST-NSs-A46, and WCEs were pulled down by glutathione beads, followed by immunoblotting with an anti-TRIM21 antibody. (D) HEK293T cells were transfected with GST-NSs-WT, GST-NSs-A46, TRIM21-V5, and p62-Myc, and WCEs were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. (E) HeLa cells were transfected with NSs-A46-3×Flag-GFP, TRIM21-V5, and p62-Myc. The cells were fixed and stained with primary antibodies (rabbit anti-V5 or mouse anti-Myc) and with secondary antibodies (Alexa Fluor 568-conjugated anti-rabbit IgG or Alexa Fluor 350-conjugated mouse IgG) for confocal microscopy. No nucleus staining was performed. (F) HEK293T cells were transfected with NSs-WT-3×Flag, NSs-A46-3×Flag, p62-HA, p62-Myc, and TRIM21-V5, and WCEs were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody.
Figure Legend Snippet: TRIM21-binding-deficient NSs mutant. (A) Schematic diagram of the NSs-A46 mutant carrying alanine substitutions at K 226 KTDG 230 . (B) HEK293T cells were transfected with NSs-WT-3×Flag, NSs-A46-3×Flag, and TRIM21-V5, and WCEs were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. (C) HEK293T cells were transfected with pEBG-GST, GST-NSs-WT, and GST-NSs-A46, and WCEs were pulled down by glutathione beads, followed by immunoblotting with an anti-TRIM21 antibody. (D) HEK293T cells were transfected with GST-NSs-WT, GST-NSs-A46, TRIM21-V5, and p62-Myc, and WCEs were immunoprecipitated with an anti-V5 antibody, followed by immunoblotting with the indicated antibody. (E) HeLa cells were transfected with NSs-A46-3×Flag-GFP, TRIM21-V5, and p62-Myc. The cells were fixed and stained with primary antibodies (rabbit anti-V5 or mouse anti-Myc) and with secondary antibodies (Alexa Fluor 568-conjugated anti-rabbit IgG or Alexa Fluor 350-conjugated mouse IgG) for confocal microscopy. No nucleus staining was performed. (F) HEK293T cells were transfected with NSs-WT-3×Flag, NSs-A46-3×Flag, p62-HA, p62-Myc, and TRIM21-V5, and WCEs were immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with the indicated antibody.

Techniques Used: Binding Assay, Mutagenesis, Transfection, Immunoprecipitation, Staining, Confocal Microscopy

39) Product Images from "Cooperative Regulation of Campylobacter jejuni Heat-Shock Genes by HspR and HrcA"

Article Title: Cooperative Regulation of Campylobacter jejuni Heat-Shock Genes by HspR and HrcA

Journal: Microorganisms

doi: 10.3390/microorganisms8081161

Glutathion S-transferase (GST)-pull down assay of purified His-HspR incubated with GST-HrcA or GST-HspR bound to Glutathione-Sepharose slurry. Upper panel: samples collected from the column containing GST alone (lane 1), GST-HspR- (lane 2) and GST-HrcA-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE together with molecular mass ladder (lane M) and stained with Coomassie Brilliant Blue. The bands corresponding to GST (+), GST-HspR (o), GST-HrcA (x) and His-HspR (*) are indicated on the right. Lower panel: samples collected from the column containing GST alone (lane 1) and GST-HrcA- (lane 2) and GST-HspR-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE, blotted on a nylon membrane and stained with anti-His Tag antibody.
Figure Legend Snippet: Glutathion S-transferase (GST)-pull down assay of purified His-HspR incubated with GST-HrcA or GST-HspR bound to Glutathione-Sepharose slurry. Upper panel: samples collected from the column containing GST alone (lane 1), GST-HspR- (lane 2) and GST-HrcA-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE together with molecular mass ladder (lane M) and stained with Coomassie Brilliant Blue. The bands corresponding to GST (+), GST-HspR (o), GST-HrcA (x) and His-HspR (*) are indicated on the right. Lower panel: samples collected from the column containing GST alone (lane 1) and GST-HrcA- (lane 2) and GST-HspR-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE, blotted on a nylon membrane and stained with anti-His Tag antibody.

Techniques Used: Pull Down Assay, Purification, Incubation, SDS Page, Staining

40) Product Images from "Nucleosome compaction facilitates HP1γ binding to methylated H3K9"

Article Title: Nucleosome compaction facilitates HP1γ binding to methylated H3K9

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv841

Magnesium ion induces the selective binding of HP1γ to H3K9me tetra-nucleosomes. ( A ) Effect of MgCl 2 on the binding of HP1γ to H3K9me3 tetra-nucleosomes. Unmethylated (unme) or H3K9me3 tetra-nucleosomes (K9me3) were incubated with GST-HP1γ (HP1γ) or GST-HP1α (HP1α) bound to GSH-Sepharose in the presence of 3 mM MgCl 2 , 50 mM NaCl. Unbound (U) and bound nucleosomes (B) are visualized (left panel), and the nucleosome core histones indicated by brackets were quantitated as in Figure 1C . Relative amounts of core histones in the bound fraction per input (%) were calculated and shown as mean ± S.E. ( n = 3) (right panel). ( B ) MgCl 2 concentration-dependent selective binding of HP1γ to H3K9me3 tetra-nucleosomes. The binding activity of HP1γ to unmethylated H3 (open circles) or H3K9me3 tetra-nucleosomes (closed circles) was determined and shown as mean ± S.E. ( n = 3) under the indicated concentration of MgCl 2 in the presence of 50 mM NaCl by pull-down assay as described in Figure 1C . ( C ) MgCl 2 concentration-dependent binding of HP1γ to native oligo-nucleosomes with H3K9me3 modification. GST-HP1γ (HP1γ) or GST (GST) bound to GSH-Sepharose was mixed with native oligo-nucleosomes and then pulled down under the condition including 50 mM NaCl, in the absence or presence of 3 mM MgCl 2 . The H3K9me3 and histone H4 in the unbound (U) and bound fractions (B) were analyzed as in Figure 1A (left panel). The amounts of H3K9me3 in the HP1γ-bound fractions over the input (%) are shown as mean ± S.E. ( n = 3) (right panel). ( D ) Schematic illustration of reconstituted mono-nucleosomes. The position of the nucleosomes on DNA is indicated by ellipses. Length (bp) of linker and nucleosome core region DNA are shown below. ( E ) Effect of MgCl 2 on the binding of HP1γ to H3K9me3 mono-nucleosomes. The binding of ummethylated H3 and H3K9me3 mono-nucleosomes (4 pmol of nucleosome core particle) to GST-HP1γ (80 pmol) bound to GSH-Sepharose in 10 μl of the reaction mixture was examined as in Figure 1C , with 50 mM NaCl, in the absence or presence of 3 mM MgCl 2 (left panel). The binding activity of HP1γ to tetra-nucleosomes reconstituted with unmethylated H3 (open circles) or H3K9me3 (closed circles) was determined and shown as mean ± S.E. ( n = 3) under the indicated concentration of MgCl 2 by pull-down assay as described in Figure 1C .
Figure Legend Snippet: Magnesium ion induces the selective binding of HP1γ to H3K9me tetra-nucleosomes. ( A ) Effect of MgCl 2 on the binding of HP1γ to H3K9me3 tetra-nucleosomes. Unmethylated (unme) or H3K9me3 tetra-nucleosomes (K9me3) were incubated with GST-HP1γ (HP1γ) or GST-HP1α (HP1α) bound to GSH-Sepharose in the presence of 3 mM MgCl 2 , 50 mM NaCl. Unbound (U) and bound nucleosomes (B) are visualized (left panel), and the nucleosome core histones indicated by brackets were quantitated as in Figure 1C . Relative amounts of core histones in the bound fraction per input (%) were calculated and shown as mean ± S.E. ( n = 3) (right panel). ( B ) MgCl 2 concentration-dependent selective binding of HP1γ to H3K9me3 tetra-nucleosomes. The binding activity of HP1γ to unmethylated H3 (open circles) or H3K9me3 tetra-nucleosomes (closed circles) was determined and shown as mean ± S.E. ( n = 3) under the indicated concentration of MgCl 2 in the presence of 50 mM NaCl by pull-down assay as described in Figure 1C . ( C ) MgCl 2 concentration-dependent binding of HP1γ to native oligo-nucleosomes with H3K9me3 modification. GST-HP1γ (HP1γ) or GST (GST) bound to GSH-Sepharose was mixed with native oligo-nucleosomes and then pulled down under the condition including 50 mM NaCl, in the absence or presence of 3 mM MgCl 2 . The H3K9me3 and histone H4 in the unbound (U) and bound fractions (B) were analyzed as in Figure 1A (left panel). The amounts of H3K9me3 in the HP1γ-bound fractions over the input (%) are shown as mean ± S.E. ( n = 3) (right panel). ( D ) Schematic illustration of reconstituted mono-nucleosomes. The position of the nucleosomes on DNA is indicated by ellipses. Length (bp) of linker and nucleosome core region DNA are shown below. ( E ) Effect of MgCl 2 on the binding of HP1γ to H3K9me3 mono-nucleosomes. The binding of ummethylated H3 and H3K9me3 mono-nucleosomes (4 pmol of nucleosome core particle) to GST-HP1γ (80 pmol) bound to GSH-Sepharose in 10 μl of the reaction mixture was examined as in Figure 1C , with 50 mM NaCl, in the absence or presence of 3 mM MgCl 2 (left panel). The binding activity of HP1γ to tetra-nucleosomes reconstituted with unmethylated H3 (open circles) or H3K9me3 (closed circles) was determined and shown as mean ± S.E. ( n = 3) under the indicated concentration of MgCl 2 by pull-down assay as described in Figure 1C .

Techniques Used: Binding Assay, Incubation, Concentration Assay, Activity Assay, Pull Down Assay, Modification

Distinct binding of HP1γ and HP1α to extended nucleosomes. ( A ) HP1γ binding to native oligo-nucleosomes prepared from HeLa cells. GSH-Sepharose bound with GST-HP1γ (HP1γ), GST-HP1α (HP1α), or GST (GST) was mixed with native oligo-nucleosomes and then pulled down. The unbound (U) and bound fractions (B) were subjected to SDS-PAGE, and H3K9me3 and histone H4 in the fractions were detected with anti-H3K9me3 (H3K9me3) and anti-H4 antibodies (H4) (left panel). Western blot data is taken from the whole gel image shown in Supplementary Figure S13. The amounts of H3K9me3 in the bound fractions over input (%) are shown as mean ± S. E. ( n = 3) (right panel). ( B ) Schematic illustration of reconstituted tetra-nucleosomes. The position of the nucleosomes on DNA is indicated by ellipses. Length (bp) of linker and nucleosome core region DNA are shown below. ( C ) Binding of GST-HP1 to the reconstituted tetra-nucleosomes. Indicated amounts of unmethylated or H3K9me3 tetra-nucleosomes (nucl) were incubated with GST-HP1γ (HP1γ) or GST-HP1α (HP1α) bound to GSH-Sepharose. Unbound (U) and bound nucleosomes (B) were separated in an 18% SDS polyacrylamide gel, stained (left panel), and positions of core histones are indicated. The amounts of core histones were quantitated, and the relative amount of core histones with unmethylated H3 (open columns) or H3K9me3 (closed columns) in the bound fraction over input (%) were calculated and shown as mean ± S.E. ( n = 3) (right panel). The whole gel for the pull-down assay is shown in Supplementary Figure S13. ( D ) The interaction of HP1γ (left panel) or HP1α (right panel) with unmodified H3 (unme) or H3K9me3 tetra-nucleosomes (K9me3) was analyzed by sucrose density gradient (15–40% (w/v) sucrose). Fractions were collected from the bottom of the tubes (B) and the proteins were separated in 18% polyacrylamide gels. HP1γ (arrow heads), HP1α (arrows), and core histones (brackets) were stained with Lumitein. ( E ) The amounts of HP1γ and HP1α in nucleosome fractions (underlined) were densitometrically determined from three independent centrifugation experiments, and the ratios to those of core histones in nucleosome fractions were calculated and are shown. The values are mean ± S.E. ( n = 3). ( F ) Schematic illustration of the chimeric HP1 proteins used in the present study. ( G ) Nucleosome binding of chimeric HP1. The binding activity was analyzed as in panel (C). The core histones and the gels were shown in the left panel, and the core histones in bound fraction over input are shown as mean ± S.E. ( n = 3) in the right panel. Asterisks indicate the position of degraded products of GST-HP1.
Figure Legend Snippet: Distinct binding of HP1γ and HP1α to extended nucleosomes. ( A ) HP1γ binding to native oligo-nucleosomes prepared from HeLa cells. GSH-Sepharose bound with GST-HP1γ (HP1γ), GST-HP1α (HP1α), or GST (GST) was mixed with native oligo-nucleosomes and then pulled down. The unbound (U) and bound fractions (B) were subjected to SDS-PAGE, and H3K9me3 and histone H4 in the fractions were detected with anti-H3K9me3 (H3K9me3) and anti-H4 antibodies (H4) (left panel). Western blot data is taken from the whole gel image shown in Supplementary Figure S13. The amounts of H3K9me3 in the bound fractions over input (%) are shown as mean ± S. E. ( n = 3) (right panel). ( B ) Schematic illustration of reconstituted tetra-nucleosomes. The position of the nucleosomes on DNA is indicated by ellipses. Length (bp) of linker and nucleosome core region DNA are shown below. ( C ) Binding of GST-HP1 to the reconstituted tetra-nucleosomes. Indicated amounts of unmethylated or H3K9me3 tetra-nucleosomes (nucl) were incubated with GST-HP1γ (HP1γ) or GST-HP1α (HP1α) bound to GSH-Sepharose. Unbound (U) and bound nucleosomes (B) were separated in an 18% SDS polyacrylamide gel, stained (left panel), and positions of core histones are indicated. The amounts of core histones were quantitated, and the relative amount of core histones with unmethylated H3 (open columns) or H3K9me3 (closed columns) in the bound fraction over input (%) were calculated and shown as mean ± S.E. ( n = 3) (right panel). The whole gel for the pull-down assay is shown in Supplementary Figure S13. ( D ) The interaction of HP1γ (left panel) or HP1α (right panel) with unmodified H3 (unme) or H3K9me3 tetra-nucleosomes (K9me3) was analyzed by sucrose density gradient (15–40% (w/v) sucrose). Fractions were collected from the bottom of the tubes (B) and the proteins were separated in 18% polyacrylamide gels. HP1γ (arrow heads), HP1α (arrows), and core histones (brackets) were stained with Lumitein. ( E ) The amounts of HP1γ and HP1α in nucleosome fractions (underlined) were densitometrically determined from three independent centrifugation experiments, and the ratios to those of core histones in nucleosome fractions were calculated and are shown. The values are mean ± S.E. ( n = 3). ( F ) Schematic illustration of the chimeric HP1 proteins used in the present study. ( G ) Nucleosome binding of chimeric HP1. The binding activity was analyzed as in panel (C). The core histones and the gels were shown in the left panel, and the core histones in bound fraction over input are shown as mean ± S.E. ( n = 3) in the right panel. Asterisks indicate the position of degraded products of GST-HP1.

Techniques Used: Binding Assay, SDS Page, Western Blot, Incubation, Staining, Pull Down Assay, Centrifugation, Activity Assay

Related Articles

MTT Assay:

Article Title: Purification and characterization of a novel plant lectin from Pinellia ternata with antineoplastic activity
Article Snippet: .. Materials The Pinellia ternata purchased from Jinmen Hubei province had been identified for its authenticity by Keli Chen in Chinese Medicine of Hubei College of traditional Chinese Medicine; PHE Sepharose Cl-4B and DEAE Sepharose Fast Flow were purchased from Amersham Pharmacia Biotech; Roswell Park Memorial Institute 1640 (RPMI 1640) was from HyClone (USA); CTX was purchased from Wuhan University Zhongnan Hospital; MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Dulbecco’s Modified Eagle’s Medium (DMEM) were products of Sino-American Biotechnology Company. .. Kunming Mice were purchased from Laboratory Animal Center of School of Medicine in Wuhan University and the experiments in vivo were performed in the same center.

Flow Cytometry:

Article Title: Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein
Article Snippet: .. Purification of recombinant HP-NAP by DEAE negative mode batch chromatography The soluble protein fraction of E . coli expressing HP-NAP at pH 9.0 and those adjusted to pH 8.5, 8.0, 7.5, and 7.0 were subjected to a small-scale DEAE negative mode batch chromatography with DEAE Sephadex A-25 (Sigma-Aldrich, St. Louis, MO, USA) and DEAE Sepharose fast flow (Amersham Pharmacia Biotech, Uppsala, Sweden) resins at their respective pH values as previously described [ ] except that the purification was performed at 25°C. .. The protein concentration of the soluble protein fraction was 0.3 mg/ml.

Article Title: Purification and characterization of a novel plant lectin from Pinellia ternata with antineoplastic activity
Article Snippet: .. Materials The Pinellia ternata purchased from Jinmen Hubei province had been identified for its authenticity by Keli Chen in Chinese Medicine of Hubei College of traditional Chinese Medicine; PHE Sepharose Cl-4B and DEAE Sepharose Fast Flow were purchased from Amersham Pharmacia Biotech; Roswell Park Memorial Institute 1640 (RPMI 1640) was from HyClone (USA); CTX was purchased from Wuhan University Zhongnan Hospital; MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Dulbecco’s Modified Eagle’s Medium (DMEM) were products of Sino-American Biotechnology Company. .. Kunming Mice were purchased from Laboratory Animal Center of School of Medicine in Wuhan University and the experiments in vivo were performed in the same center.

Article Title: Pre-mRNA 3’ Cleavage is Reversibly Inhibited In Vitro by Cleavage Factor Dephosphorylation
Article Snippet: .. For fractionation, nuclear extract containing approximately 75 mg total protein was loaded onto a 19 ml hand-packed DEAE-Sepharose Fast Flow (GE Healthcare) column and eluted as previously described. .. Active fractions were identified either by in vitro polyadenylation (CPSF, fractions supplemented with recombinant bovine PAP II), or by in vitro cleavage (CFm ) using small amounts of previously purified CPSF and CstF.

Article Title: Dacin, one metalloproteinase from Deinagkistrodon acutus venom inhibiting contraction of mouse ileum muscle
Article Snippet: .. Reagents Sephadex G-50, DEAE Sepharose Fast Flow and Hitrap Capto DEAE were purchased from GE Healthcare (USA). .. Protein MW Marker (Low) was obtained from TAKARA (Japan), ACN and Methanol from Fulltime Co. (China), and Bovine thrombin and fibrinogen from Biosharp (China).

Ion Exchange Chromatography:

Article Title: Crystallization and preliminary X-ray crystallographic analysis of agkicetin-C from Deinagkistrodon acutus venom
Article Snippet: .. 1 g of crude D. acutus venom (purchased from Huangshan Institute of Snakes, Anhui, China) was first fractioned by ion-exchange chromatography on DEAE-Sepharose (Amersham-Pharmacia, Uppsala, Sweden) and agkicetin-C was then purified by a minor modification of Chen’s method (Chen & Tsai, 1995 ) (Fig. 1 ). ..

Chromatography:

Article Title: Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein
Article Snippet: .. Purification of recombinant HP-NAP by DEAE negative mode batch chromatography The soluble protein fraction of E . coli expressing HP-NAP at pH 9.0 and those adjusted to pH 8.5, 8.0, 7.5, and 7.0 were subjected to a small-scale DEAE negative mode batch chromatography with DEAE Sephadex A-25 (Sigma-Aldrich, St. Louis, MO, USA) and DEAE Sepharose fast flow (Amersham Pharmacia Biotech, Uppsala, Sweden) resins at their respective pH values as previously described [ ] except that the purification was performed at 25°C. .. The protein concentration of the soluble protein fraction was 0.3 mg/ml.

Purification:

Article Title: Crystallization and preliminary X-ray crystallographic analysis of agkicetin-C from Deinagkistrodon acutus venom
Article Snippet: .. 1 g of crude D. acutus venom (purchased from Huangshan Institute of Snakes, Anhui, China) was first fractioned by ion-exchange chromatography on DEAE-Sepharose (Amersham-Pharmacia, Uppsala, Sweden) and agkicetin-C was then purified by a minor modification of Chen’s method (Chen & Tsai, 1995 ) (Fig. 1 ). ..

Article Title: Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein
Article Snippet: .. Purification of recombinant HP-NAP by DEAE negative mode batch chromatography The soluble protein fraction of E . coli expressing HP-NAP at pH 9.0 and those adjusted to pH 8.5, 8.0, 7.5, and 7.0 were subjected to a small-scale DEAE negative mode batch chromatography with DEAE Sephadex A-25 (Sigma-Aldrich, St. Louis, MO, USA) and DEAE Sepharose fast flow (Amersham Pharmacia Biotech, Uppsala, Sweden) resins at their respective pH values as previously described [ ] except that the purification was performed at 25°C. .. The protein concentration of the soluble protein fraction was 0.3 mg/ml.

other:

Article Title: Structural Interactions in Chondroitin 4-Sulfate-mediated Adherence of Plasmodium falciparum-infected Erythrocytes in Human Placenta During Pregnancy-Associated Malaria †
Article Snippet: DEAE-Sepharose and PD-10 columns were from Amersham Pharmacia; Bio-Gel P-4 and Bio-Gel P-6 were from Bio-Rad; Polystyrene Petri dishes (Falcon 1058) were from Becton-Dickinson Labware.

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid
Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Fractionation:

Article Title: Pre-mRNA 3’ Cleavage is Reversibly Inhibited In Vitro by Cleavage Factor Dephosphorylation
Article Snippet: .. For fractionation, nuclear extract containing approximately 75 mg total protein was loaded onto a 19 ml hand-packed DEAE-Sepharose Fast Flow (GE Healthcare) column and eluted as previously described. .. Active fractions were identified either by in vitro polyadenylation (CPSF, fractions supplemented with recombinant bovine PAP II), or by in vitro cleavage (CFm ) using small amounts of previously purified CPSF and CstF.

Expressing:

Article Title: Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein
Article Snippet: .. Purification of recombinant HP-NAP by DEAE negative mode batch chromatography The soluble protein fraction of E . coli expressing HP-NAP at pH 9.0 and those adjusted to pH 8.5, 8.0, 7.5, and 7.0 were subjected to a small-scale DEAE negative mode batch chromatography with DEAE Sephadex A-25 (Sigma-Aldrich, St. Louis, MO, USA) and DEAE Sepharose fast flow (Amersham Pharmacia Biotech, Uppsala, Sweden) resins at their respective pH values as previously described [ ] except that the purification was performed at 25°C. .. The protein concentration of the soluble protein fraction was 0.3 mg/ml.

Modification:

Article Title: Crystallization and preliminary X-ray crystallographic analysis of agkicetin-C from Deinagkistrodon acutus venom
Article Snippet: .. 1 g of crude D. acutus venom (purchased from Huangshan Institute of Snakes, Anhui, China) was first fractioned by ion-exchange chromatography on DEAE-Sepharose (Amersham-Pharmacia, Uppsala, Sweden) and agkicetin-C was then purified by a minor modification of Chen’s method (Chen & Tsai, 1995 ) (Fig. 1 ). ..

Article Title: Purification and characterization of a novel plant lectin from Pinellia ternata with antineoplastic activity
Article Snippet: .. Materials The Pinellia ternata purchased from Jinmen Hubei province had been identified for its authenticity by Keli Chen in Chinese Medicine of Hubei College of traditional Chinese Medicine; PHE Sepharose Cl-4B and DEAE Sepharose Fast Flow were purchased from Amersham Pharmacia Biotech; Roswell Park Memorial Institute 1640 (RPMI 1640) was from HyClone (USA); CTX was purchased from Wuhan University Zhongnan Hospital; MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Dulbecco’s Modified Eagle’s Medium (DMEM) were products of Sino-American Biotechnology Company. .. Kunming Mice were purchased from Laboratory Animal Center of School of Medicine in Wuhan University and the experiments in vivo were performed in the same center.

Recombinant:

Article Title: Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein
Article Snippet: .. Purification of recombinant HP-NAP by DEAE negative mode batch chromatography The soluble protein fraction of E . coli expressing HP-NAP at pH 9.0 and those adjusted to pH 8.5, 8.0, 7.5, and 7.0 were subjected to a small-scale DEAE negative mode batch chromatography with DEAE Sephadex A-25 (Sigma-Aldrich, St. Louis, MO, USA) and DEAE Sepharose fast flow (Amersham Pharmacia Biotech, Uppsala, Sweden) resins at their respective pH values as previously described [ ] except that the purification was performed at 25°C. .. The protein concentration of the soluble protein fraction was 0.3 mg/ml.

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  • 99
    GE Healthcare glutathione gsh sepharose
    The PHD2-p23 interaction facilitates prolyl hydroxylation of HIF-1α. A , HeLa cells were treated with control ( Con ) or p23 siRNA, and cellular extracts were prepared. GST or GST-HIF-1α (531–575) prebound to <t>GSH-agarose</t> was incubated
    Glutathione Gsh Sepharose, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare glutathione sepharose resin
    Glutathion S-transferase (GST)-pull down assay of purified His-HspR incubated with GST-HrcA or GST-HspR bound to <t>Glutathione-Sepharose</t> slurry. Upper panel: samples collected from the column containing GST alone (lane 1), GST-HspR- (lane 2) and GST-HrcA-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE together with molecular mass ladder (lane M) and stained with Coomassie Brilliant Blue. The bands corresponding to GST (+), GST-HspR (o), GST-HrcA (x) and His-HspR (*) are indicated on the right. Lower panel: samples collected from the column containing GST alone (lane 1) and GST-HrcA- (lane 2) and GST-HspR-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE, blotted on a nylon membrane and stained with anti-His Tag antibody.
    Glutathione Sepharose Resin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The PHD2-p23 interaction facilitates prolyl hydroxylation of HIF-1α. A , HeLa cells were treated with control ( Con ) or p23 siRNA, and cellular extracts were prepared. GST or GST-HIF-1α (531–575) prebound to GSH-agarose was incubated

    Journal: The Journal of Biological Chemistry

    Article Title: Prolyl Hydroxylase Domain Protein 2 (PHD2) Binds a Pro-Xaa-Leu-Glu Motif, Linking It to the Heat Shock Protein 90 Pathway *

    doi: 10.1074/jbc.M112.440552

    Figure Lengend Snippet: The PHD2-p23 interaction facilitates prolyl hydroxylation of HIF-1α. A , HeLa cells were treated with control ( Con ) or p23 siRNA, and cellular extracts were prepared. GST or GST-HIF-1α (531–575) prebound to GSH-agarose was incubated

    Article Snippet: GST and GST-HIF-1α (531–575) were purified from E. coli DH5α transformed with pGEX-5X-1 and pGEX-HIF-1α (531–575), respectively, using affinity chromatography on glutathione (GSH)-Sepharose (GE Healthcare).

    Techniques: Incubation

    Myo5a-HMM and the MD-IQ1 bind to GST-GTD in a Ca 2+ -dependent manner. FLAG-tagged MD-IQ1 or Myo5a-HMM was incubated with GST-GTD or GST and was pulled down by GSH-Sepharose under EGTA or pCa4 conditions. The pulled-down samples (20 μL each) were

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

    Article Title: Calmodulin in complex with the first IQ motif of myosin-5a functions as an intact calcium sensor

    doi: 10.1073/pnas.1607702113

    Figure Lengend Snippet: Myo5a-HMM and the MD-IQ1 bind to GST-GTD in a Ca 2+ -dependent manner. FLAG-tagged MD-IQ1 or Myo5a-HMM was incubated with GST-GTD or GST and was pulled down by GSH-Sepharose under EGTA or pCa4 conditions. The pulled-down samples (20 μL each) were

    Article Snippet: For the pull-down assay under EGTA conditions, 100 μL of 0.5 μM GST-GTD or GST and 1 μM FLAG-tagged MD-IQ1 or Myo5a-HMM in washing buffer [20 mM 3-(N-morpholino)propanesulfonic acid (Mops)-KOH (pH 7.0), 50 mM NaCl, 1 mM MgCl2 , 1 mM EGTA, and 1 mM DTT] were mixed with 10 μL of glutathione (GSH)-Sepharose (GE Healthcare) and incubated with rotation at 4 °C for 2 h. After three washings with 200 μL washing buffer to remove the unbound proteins, the bound proteins were eluted with 40 μL elution buffer [10 mM GSH, 50 mM Tris⋅HCl (pH 7.5), 50 mM NaCl, and 1 mM DTT].

    Techniques: Incubation

    B. pseudomallei BipC interacts with cellular actin. (A) GST-BipC interacts with actin in murine splenic lysates. GST, GST-BimA 48−384 , GST-SipC, and GST-BipC bound to GSH-linked Sepharose beads were incubated with murine splenic lysates in polymerization buffer. The Sepharose beads were washed and the bound proteins were denatured in Laemmli buffer. The Coomassie stained gel shows the relative quantities of input proteins used in the pull-down assay. Equal volumes (5 μl) of each pulldown sample (representing a half of the total samples) were analyzed by SDS-PAGE and Western blot. The α-actin Western blot indicates actin binding to the fusion proteins. (B) GST-BipC directly interacts with actin in the absence of other cellular proteins. GSH-Sepharose beads coated with either GST, GST-BimA 48−384 ,or GST-BipC were mixed with rhodamine-labeled actin suspended either in polymerization buffer or PBS and immediately imaged using a confocal microscope. The formation of a red “halo” around the bead indicates binding of actin to the bead surface. These are also shown as grayscale images for clarity. DIC/phase contrast images of the beads are also shown. Scale bar = 20 μm. ( C ) GST-BipC preferentially binds F-actin. Actin was allowed to polymerize at room temperature for 2 h before being mixed with GST, GST-BimA 48−384 , GST-SipC, or GST-BipC. The mixtures were submitted to ultra-centrifugation to separate the monomeric actin (supernatant) and the filamentous actin (pellet). Proteins in the supernatant ( S ) and pellet ( P ) fractions were separated by SDS-PAGE and visualized by silver staining. The GST-fusion proteins are indicated by the blue arrows. The average percentage and standard deviation over the three replicates of each GST-fusion protein distributed in the supernatant or pellet (as determined by densitometry) is shown below the corresponding lane in the image (shown as % ).

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity

    doi: 10.3389/fcimb.2017.00333

    Figure Lengend Snippet: B. pseudomallei BipC interacts with cellular actin. (A) GST-BipC interacts with actin in murine splenic lysates. GST, GST-BimA 48−384 , GST-SipC, and GST-BipC bound to GSH-linked Sepharose beads were incubated with murine splenic lysates in polymerization buffer. The Sepharose beads were washed and the bound proteins were denatured in Laemmli buffer. The Coomassie stained gel shows the relative quantities of input proteins used in the pull-down assay. Equal volumes (5 μl) of each pulldown sample (representing a half of the total samples) were analyzed by SDS-PAGE and Western blot. The α-actin Western blot indicates actin binding to the fusion proteins. (B) GST-BipC directly interacts with actin in the absence of other cellular proteins. GSH-Sepharose beads coated with either GST, GST-BimA 48−384 ,or GST-BipC were mixed with rhodamine-labeled actin suspended either in polymerization buffer or PBS and immediately imaged using a confocal microscope. The formation of a red “halo” around the bead indicates binding of actin to the bead surface. These are also shown as grayscale images for clarity. DIC/phase contrast images of the beads are also shown. Scale bar = 20 μm. ( C ) GST-BipC preferentially binds F-actin. Actin was allowed to polymerize at room temperature for 2 h before being mixed with GST, GST-BimA 48−384 , GST-SipC, or GST-BipC. The mixtures were submitted to ultra-centrifugation to separate the monomeric actin (supernatant) and the filamentous actin (pellet). Proteins in the supernatant ( S ) and pellet ( P ) fractions were separated by SDS-PAGE and visualized by silver staining. The GST-fusion proteins are indicated by the blue arrows. The average percentage and standard deviation over the three replicates of each GST-fusion protein distributed in the supernatant or pellet (as determined by densitometry) is shown below the corresponding lane in the image (shown as % ).

    Article Snippet: For GST, GST-BimA, and GST-BipC, glutathione (GSH)-linked Sepharose beads (GE Healthcare) were added to the bacterial lysate and incubated at 4°C for 1 h with agitation.

    Techniques: Incubation, Staining, Pull Down Assay, SDS Page, Western Blot, Binding Assay, Labeling, Microscopy, Centrifugation, Silver Staining, Standard Deviation

    Glutathion S-transferase (GST)-pull down assay of purified His-HspR incubated with GST-HrcA or GST-HspR bound to Glutathione-Sepharose slurry. Upper panel: samples collected from the column containing GST alone (lane 1), GST-HspR- (lane 2) and GST-HrcA-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE together with molecular mass ladder (lane M) and stained with Coomassie Brilliant Blue. The bands corresponding to GST (+), GST-HspR (o), GST-HrcA (x) and His-HspR (*) are indicated on the right. Lower panel: samples collected from the column containing GST alone (lane 1) and GST-HrcA- (lane 2) and GST-HspR-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE, blotted on a nylon membrane and stained with anti-His Tag antibody.

    Journal: Microorganisms

    Article Title: Cooperative Regulation of Campylobacter jejuni Heat-Shock Genes by HspR and HrcA

    doi: 10.3390/microorganisms8081161

    Figure Lengend Snippet: Glutathion S-transferase (GST)-pull down assay of purified His-HspR incubated with GST-HrcA or GST-HspR bound to Glutathione-Sepharose slurry. Upper panel: samples collected from the column containing GST alone (lane 1), GST-HspR- (lane 2) and GST-HrcA-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE together with molecular mass ladder (lane M) and stained with Coomassie Brilliant Blue. The bands corresponding to GST (+), GST-HspR (o), GST-HrcA (x) and His-HspR (*) are indicated on the right. Lower panel: samples collected from the column containing GST alone (lane 1) and GST-HrcA- (lane 2) and GST-HspR-GSH-Sepharose slurry (lane 3) were separated by SDS-PAGE, blotted on a nylon membrane and stained with anti-His Tag antibody.

    Article Snippet: The soluble protein fraction was then incubated with 100 μL of Glutathione-Sepharose resin (GSH, Glutathione Sepharose® 4B, GE Healthcare, Chicago, IL, USA) for 1 h at 4 °C.

    Techniques: Pull Down Assay, Purification, Incubation, SDS Page, Staining