Structured Review

GE Healthcare glutathione sepharose 4b beads
CaM binding sites in the i3 loop and carboxyl tail of the AT 1A receptor. A, Schematic representation of constructs of GST-fusion proteins. Four GST-fusion proteins containing truncated peptides in the i3 loop and carboxyl tail of the receptor were constructed, including GST-N-terminus of the i3 loop (GST-ATi3N), GST-C-terminus of the i3 loop (GST-ATi3C), GST-N-terminus of carboxyl tail (GST-ATctN) and GST-C-terminus of carboxyl tail (GST-ATctC). The numbers under the first or the last residues represent amino acid positions in the receptor. B, Interaction of GST-fusion proteins with CaM. The GST-fusion proteins (50 pmol) were incubated with purified bovine CaM (50 pmol) in 250 µl of buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . Proteins were pulled down by <t>gluthathione-sepharose</t> 4B beads, following which immunoblots were probed with a specific anti-CaM antibody. These experiments were repeated five times with similar results.
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1) Product Images from "Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor"

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065266

CaM binding sites in the i3 loop and carboxyl tail of the AT 1A receptor. A, Schematic representation of constructs of GST-fusion proteins. Four GST-fusion proteins containing truncated peptides in the i3 loop and carboxyl tail of the receptor were constructed, including GST-N-terminus of the i3 loop (GST-ATi3N), GST-C-terminus of the i3 loop (GST-ATi3C), GST-N-terminus of carboxyl tail (GST-ATctN) and GST-C-terminus of carboxyl tail (GST-ATctC). The numbers under the first or the last residues represent amino acid positions in the receptor. B, Interaction of GST-fusion proteins with CaM. The GST-fusion proteins (50 pmol) were incubated with purified bovine CaM (50 pmol) in 250 µl of buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . Proteins were pulled down by gluthathione-sepharose 4B beads, following which immunoblots were probed with a specific anti-CaM antibody. These experiments were repeated five times with similar results.
Figure Legend Snippet: CaM binding sites in the i3 loop and carboxyl tail of the AT 1A receptor. A, Schematic representation of constructs of GST-fusion proteins. Four GST-fusion proteins containing truncated peptides in the i3 loop and carboxyl tail of the receptor were constructed, including GST-N-terminus of the i3 loop (GST-ATi3N), GST-C-terminus of the i3 loop (GST-ATi3C), GST-N-terminus of carboxyl tail (GST-ATctN) and GST-C-terminus of carboxyl tail (GST-ATctC). The numbers under the first or the last residues represent amino acid positions in the receptor. B, Interaction of GST-fusion proteins with CaM. The GST-fusion proteins (50 pmol) were incubated with purified bovine CaM (50 pmol) in 250 µl of buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . Proteins were pulled down by gluthathione-sepharose 4B beads, following which immunoblots were probed with a specific anti-CaM antibody. These experiments were repeated five times with similar results.

Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Construct, Incubation, Purification, Western Blot

Effect of CaM on G protein βγ subunit interaction with wild ATi3 or ATct. A. Interaction of G protein βγ subunits and ATi3 or ATct. 50 pmol of GST-ATi3(213–242) or GST-ATct(297–359) were incubated with different amounts of G protein Gβγ subunits in a buffer containing 20 mM Tris-HCl and 70 mM NaCl (pH 7.5). Interactions were assessed by GST-fusion protein pull-down assay, and blots were probed with a specific antibody against Gβ subunits (upper panel). 1 pmol of Gβγ subunit was interacted with different amounts of GST-ATi3(213–242) and GST-ATct(297–359). Interaction was assessed by immunoblot against Gβ subunits (lower panel). B. CaM inhibits the interaction between G protein Gβγ subunits and ATi3 or ATct. 2 pmol of GST-ATi3(213–242) or GST-ATct(297–359) were incubated with different concentrations of pure bovine brain CaM for 30 minutes in a buffer (20 mM Tris-HCl and 70 mM NaCl with 0.1 mM CaCl 2 , pH 7.5) following which 2 pmol of Gβγ subunits were added and incubated for 1 hour. GST-fusion proteins and their interacting proteins were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot. Blots were probed with a specific antibody against Gβ subunits. The summary graph represents means ± S.E. from four independent experiments. C. Effects of CaM on the interactions between G protein βγ subunits and mutated ATi3 and ATct. The method is same as the described in the Figure 7B , except that we used mutated GST-fusion proteins, GST-ATi3(213–242)W219A, ATct(297–359)F309A, and ATct(297–359)F313A. The summary graph represents mean ± S.E. from 4 or 5 independent experiments.
Figure Legend Snippet: Effect of CaM on G protein βγ subunit interaction with wild ATi3 or ATct. A. Interaction of G protein βγ subunits and ATi3 or ATct. 50 pmol of GST-ATi3(213–242) or GST-ATct(297–359) were incubated with different amounts of G protein Gβγ subunits in a buffer containing 20 mM Tris-HCl and 70 mM NaCl (pH 7.5). Interactions were assessed by GST-fusion protein pull-down assay, and blots were probed with a specific antibody against Gβ subunits (upper panel). 1 pmol of Gβγ subunit was interacted with different amounts of GST-ATi3(213–242) and GST-ATct(297–359). Interaction was assessed by immunoblot against Gβ subunits (lower panel). B. CaM inhibits the interaction between G protein Gβγ subunits and ATi3 or ATct. 2 pmol of GST-ATi3(213–242) or GST-ATct(297–359) were incubated with different concentrations of pure bovine brain CaM for 30 minutes in a buffer (20 mM Tris-HCl and 70 mM NaCl with 0.1 mM CaCl 2 , pH 7.5) following which 2 pmol of Gβγ subunits were added and incubated for 1 hour. GST-fusion proteins and their interacting proteins were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot. Blots were probed with a specific antibody against Gβ subunits. The summary graph represents means ± S.E. from four independent experiments. C. Effects of CaM on the interactions between G protein βγ subunits and mutated ATi3 and ATct. The method is same as the described in the Figure 7B , except that we used mutated GST-fusion proteins, GST-ATi3(213–242)W219A, ATct(297–359)F309A, and ATct(297–359)F313A. The summary graph represents mean ± S.E. from 4 or 5 independent experiments.

Techniques Used: Chick Chorioallantoic Membrane Assay, Incubation, Pull Down Assay

Interactions of the i3 loop and carboxyl tail of the AT 1A receptor with CaM. A, Construction of GST-fusion proteins. Upper panel illustrates a schematic structure of the AT 1A receptor and constructs of GST-fusion proteins of the i3 loop and carboxyl tail of the receptor. The numbers represent positions of amino acids in the receptor. The GST-fusion proteins were constructed, expressed in E. coli , purified by using gluthathione-sepharose 4B beads, separated by SDS-PAGE, and stained by Coomassie blue (lower panel). B. Interactions of the i3 loop or carboxyl tail of AT 1A receptor with CaM in rat brain lysates (upper panel) and with purified bovine brain CaM (lower panel). 50 pmol of GST-fusion proteins were incubated with 500 µg of rat brain lysates in 250 µl of buffer containing 20 mM Tris-HCl, 70 mM NaCl, pH 7.5 with 1 mM EGTA or 0.1 mM CaCl 2 . Interacting protein complexes were pulled down by gluthathione-sepharose 4B beads, and visualized by immunoblot with a specific anti-CaM antibody. The same methods were applied to interactions with pure bovine CaM (50 pmol), except that the buffer contained 100 mM Tris-HCl (pH 7.5). These experiments were repeated five times with similar results.
Figure Legend Snippet: Interactions of the i3 loop and carboxyl tail of the AT 1A receptor with CaM. A, Construction of GST-fusion proteins. Upper panel illustrates a schematic structure of the AT 1A receptor and constructs of GST-fusion proteins of the i3 loop and carboxyl tail of the receptor. The numbers represent positions of amino acids in the receptor. The GST-fusion proteins were constructed, expressed in E. coli , purified by using gluthathione-sepharose 4B beads, separated by SDS-PAGE, and stained by Coomassie blue (lower panel). B. Interactions of the i3 loop or carboxyl tail of AT 1A receptor with CaM in rat brain lysates (upper panel) and with purified bovine brain CaM (lower panel). 50 pmol of GST-fusion proteins were incubated with 500 µg of rat brain lysates in 250 µl of buffer containing 20 mM Tris-HCl, 70 mM NaCl, pH 7.5 with 1 mM EGTA or 0.1 mM CaCl 2 . Interacting protein complexes were pulled down by gluthathione-sepharose 4B beads, and visualized by immunoblot with a specific anti-CaM antibody. The same methods were applied to interactions with pure bovine CaM (50 pmol), except that the buffer contained 100 mM Tris-HCl (pH 7.5). These experiments were repeated five times with similar results.

Techniques Used: Chick Chorioallantoic Membrane Assay, Construct, Purification, SDS Page, Staining, Incubation

Effect of point mutations in ATi3N or ATctN on their interactions with CaM. A. Modeled structures of CaM-ATi3(214–231) and CaM-ATct(302–317). The complexes of CaM and CaM binding motif in the i3 loop ATi3(214–231) or the carboxyl terminal tail ATct(302–317) of the receptor were modeled as described in Experimental Procedures. The target peptides are colored in red. Residues W219 in ATi3(214–231)−SYTLIWKALKKAYEIQKN, and F309 and F313 in ATct(302–317)−YGFLGKKFKKYFLQLL are displayed with sticks and are colored in blue. Calcium atoms are shown as orange spheres. The N- and C- termini of CaM are also labeled. Helices and sheets in CaM are colored in green and yellow, respectively. B. Effect of point mutations at ATi3N or ATctN on their interaction with CaM. 50 pmol of wild type GST-fusion proteins including GST-ATi3N(213–234) and GST-ATctN (297–324), and 50 pmol of mutated GST-fusion proteins including GST-ATi3N(W219A), GST-ATctN(F309A) and GST-ATctN(F313A), were incubated with purified bovine brain CaM in a buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . The protein complexes were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot with a specific anti-CaM antibody. GST-fusion proteins were visualized in the gels by Coomassie blue staining (the lower gel panel). The summary graph represents relative densities of the ratio of the CaM in the immunoblots and the loaded GST-fusion proteins as determined by Coomassie blue staining. The bars represent mean ± S.E. from 5 independent experiments. * or # stand for P
Figure Legend Snippet: Effect of point mutations in ATi3N or ATctN on their interactions with CaM. A. Modeled structures of CaM-ATi3(214–231) and CaM-ATct(302–317). The complexes of CaM and CaM binding motif in the i3 loop ATi3(214–231) or the carboxyl terminal tail ATct(302–317) of the receptor were modeled as described in Experimental Procedures. The target peptides are colored in red. Residues W219 in ATi3(214–231)−SYTLIWKALKKAYEIQKN, and F309 and F313 in ATct(302–317)−YGFLGKKFKKYFLQLL are displayed with sticks and are colored in blue. Calcium atoms are shown as orange spheres. The N- and C- termini of CaM are also labeled. Helices and sheets in CaM are colored in green and yellow, respectively. B. Effect of point mutations at ATi3N or ATctN on their interaction with CaM. 50 pmol of wild type GST-fusion proteins including GST-ATi3N(213–234) and GST-ATctN (297–324), and 50 pmol of mutated GST-fusion proteins including GST-ATi3N(W219A), GST-ATctN(F309A) and GST-ATctN(F313A), were incubated with purified bovine brain CaM in a buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . The protein complexes were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot with a specific anti-CaM antibody. GST-fusion proteins were visualized in the gels by Coomassie blue staining (the lower gel panel). The summary graph represents relative densities of the ratio of the CaM in the immunoblots and the loaded GST-fusion proteins as determined by Coomassie blue staining. The bars represent mean ± S.E. from 5 independent experiments. * or # stand for P

Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Labeling, Incubation, Purification, Staining, Western Blot

2) Product Images from "Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1–Mad2"

Article Title: Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1–Mad2

Journal: Nature Communications

doi: 10.1038/ncomms10151

Cep57 interacts with Mad1. ( a ) Mitotic HeLa cells arrested by nocodazole were used for immunoprecipitation (IP) with anti-Cep57 antibody and western blotting with anti-Cep57 and anti-Mad1 antibodies. IgG served as the negative control. ( b ) HEK293T cells were co-transfected with the indicated plasmids, and were used to for IP and western blotting. ( c ) Binding assays of Mad1 and Cep57. Flag-Cep57 and Mad1-GFP (expressed in HEK293T cells and purified) were incubated for IP with anti-GFP antibody. IP samples were analysed by western blotting with anti-Flag and anti-GFP antibodies. ( d ) In vitro pull-down assays of Mad1 and Cep57. GST-Mad1 (176–718 amino acids) and MBP-Cep57 (151–500 amino acids) (expressed in E. coli and purified) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by western blotting with anti-GST antibody and Coomassie blue staining. ( e ) GST pull-down assays of Cep57 and Mad1. Lysates of HEK293T cells overexpressing Mad1-GFP were incubated with Glutathione Sepharose 4B beads coated with GST, GST-Cep57 (1–242 amino acids) or GST-Cep57 (195–500 amino acids). The samples were analysed by western blotting with anti-GFP antibody. GST and GST-tagged proteins were stained with Coomassie blue. ( f ) Binding assays of Mad1 and Cep57 (195–500 amino acids). GST-Cep57 (195–500 amino acids; expressed in E. coli ) and Flag-Mad1 (expressed in HEK293T cells) were purified and incubated with anti-Flag antibody. The IP samples with anti-Flag antibody were analysed by western blotting with anti-GST and anti-Flag antibodies. ( g ) Schematic of truncated mutants of Mad1. ( h , i ) IP using lysates of HEK293T cells co-overexpressing GFP-Cep57 and Flag-Mad1 (FL, 1–530 and 531–718 amino acids) ( h ) or Flag-Mad1 (1–530, 1–175, 176–350 and 351–530 amino acids) ( i ) with anti-Flag antibody. The IP samples were analysed by western blotting with anti-GFP and anti-Flag antibodies. FL, full length. ( j ) Quantification and normalization of the kinetochore signal of Flag-Mad1 in HeLa cells that were transfected with FL and truncated mutants of Flag-Mad1 and treated with nocodazole for 1 h. Greater than 50 kinetochores from 5 cells were measured. The experiment was repeated three times. Data are mean±s.e.m.
Figure Legend Snippet: Cep57 interacts with Mad1. ( a ) Mitotic HeLa cells arrested by nocodazole were used for immunoprecipitation (IP) with anti-Cep57 antibody and western blotting with anti-Cep57 and anti-Mad1 antibodies. IgG served as the negative control. ( b ) HEK293T cells were co-transfected with the indicated plasmids, and were used to for IP and western blotting. ( c ) Binding assays of Mad1 and Cep57. Flag-Cep57 and Mad1-GFP (expressed in HEK293T cells and purified) were incubated for IP with anti-GFP antibody. IP samples were analysed by western blotting with anti-Flag and anti-GFP antibodies. ( d ) In vitro pull-down assays of Mad1 and Cep57. GST-Mad1 (176–718 amino acids) and MBP-Cep57 (151–500 amino acids) (expressed in E. coli and purified) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by western blotting with anti-GST antibody and Coomassie blue staining. ( e ) GST pull-down assays of Cep57 and Mad1. Lysates of HEK293T cells overexpressing Mad1-GFP were incubated with Glutathione Sepharose 4B beads coated with GST, GST-Cep57 (1–242 amino acids) or GST-Cep57 (195–500 amino acids). The samples were analysed by western blotting with anti-GFP antibody. GST and GST-tagged proteins were stained with Coomassie blue. ( f ) Binding assays of Mad1 and Cep57 (195–500 amino acids). GST-Cep57 (195–500 amino acids; expressed in E. coli ) and Flag-Mad1 (expressed in HEK293T cells) were purified and incubated with anti-Flag antibody. The IP samples with anti-Flag antibody were analysed by western blotting with anti-GST and anti-Flag antibodies. ( g ) Schematic of truncated mutants of Mad1. ( h , i ) IP using lysates of HEK293T cells co-overexpressing GFP-Cep57 and Flag-Mad1 (FL, 1–530 and 531–718 amino acids) ( h ) or Flag-Mad1 (1–530, 1–175, 176–350 and 351–530 amino acids) ( i ) with anti-Flag antibody. The IP samples were analysed by western blotting with anti-GFP and anti-Flag antibodies. FL, full length. ( j ) Quantification and normalization of the kinetochore signal of Flag-Mad1 in HeLa cells that were transfected with FL and truncated mutants of Flag-Mad1 and treated with nocodazole for 1 h. Greater than 50 kinetochores from 5 cells were measured. The experiment was repeated three times. Data are mean±s.e.m.

Techniques Used: Immunoprecipitation, Western Blot, Negative Control, Transfection, Binding Assay, Purification, Incubation, In Vitro, Magnetic Beads, Staining

Cep57 is a novel kinetochore component in human cells. ( a ) Three-dimensional structured illumination microscopy (SIM) images of HeLa cells double-immunostained with antibodies against Cep57 (green) and CREST (red). ( b ) Immunofluorescence of Cep57 (green) and CENP-A (red) in HeLa cells. ( c ) Immunofluorescence of Cep57 (green) and Mis12 (red) in HeLa cells at metaphase after treatment with MG132 for 1 h. A linescan through the kinetochore pair indicates the co-localization of Cep57 with Mis12. ( d ) Stimulated emission depletion (STED) images of immunofluorescence of Cep57 (green) and Zwint-1 (red) in RPE1 cells. ( e ) Immunofluorescence of Cep57 (green) and CREST (red) in HeLa cells at different stages during mitosis. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). ( f ) HEK293T cells were co-transfected with Cep57-GFP and Mis12-HA. The cell lysates were immunoprecipitated (IP) and analysed by western blotting (WB) with the indicated antibodies. ( g ) Binding assays of Mis12 and Cep57. GST-Mis12 (expressed in E. coli and purified) was incubated with Flag-Cep57 (expressed in HEK293T cells and purified). The IP samples with anti-Flag antibody were analysed by WB with anti-Flag and anti-Mis12 antibodies. ( h ) Schematic of Cep57 truncated mutants. ( i ) GST pull-down assays of Cep57 and Mis12. Lysates of HEK293T cells overexpressing Mis12-HA were incubated with Glutathione Sepharose 4B beads coated with GST or GST-Cep57N/C. The samples were analysed by WB with anti-HA antibody. GST-tagged proteins were stained with Coomassie blue. ( j ) In vitro pull-down assays of Mis12 and Cep57 (1–242 amino acids). GST-Mis12 (expressed in E. coli and purified) and MBP-Cep57 (1–242 amino acids) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by WB with anti-GST antibody and Coomassie blue staining. ( k ) Quantification of kinetochore signals of Cep57 and Mis12 in HeLa cells depleted of Cep57, Mis12 or negative control (NC) by siRNAs. The signal from control siRNA-treated cells was normalized to 1.0. More than 200 kinetochores from 20 cells were measured. The experiment was repeated three times. Data are mean±s.e.m. **** P
Figure Legend Snippet: Cep57 is a novel kinetochore component in human cells. ( a ) Three-dimensional structured illumination microscopy (SIM) images of HeLa cells double-immunostained with antibodies against Cep57 (green) and CREST (red). ( b ) Immunofluorescence of Cep57 (green) and CENP-A (red) in HeLa cells. ( c ) Immunofluorescence of Cep57 (green) and Mis12 (red) in HeLa cells at metaphase after treatment with MG132 for 1 h. A linescan through the kinetochore pair indicates the co-localization of Cep57 with Mis12. ( d ) Stimulated emission depletion (STED) images of immunofluorescence of Cep57 (green) and Zwint-1 (red) in RPE1 cells. ( e ) Immunofluorescence of Cep57 (green) and CREST (red) in HeLa cells at different stages during mitosis. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). ( f ) HEK293T cells were co-transfected with Cep57-GFP and Mis12-HA. The cell lysates were immunoprecipitated (IP) and analysed by western blotting (WB) with the indicated antibodies. ( g ) Binding assays of Mis12 and Cep57. GST-Mis12 (expressed in E. coli and purified) was incubated with Flag-Cep57 (expressed in HEK293T cells and purified). The IP samples with anti-Flag antibody were analysed by WB with anti-Flag and anti-Mis12 antibodies. ( h ) Schematic of Cep57 truncated mutants. ( i ) GST pull-down assays of Cep57 and Mis12. Lysates of HEK293T cells overexpressing Mis12-HA were incubated with Glutathione Sepharose 4B beads coated with GST or GST-Cep57N/C. The samples were analysed by WB with anti-HA antibody. GST-tagged proteins were stained with Coomassie blue. ( j ) In vitro pull-down assays of Mis12 and Cep57 (1–242 amino acids). GST-Mis12 (expressed in E. coli and purified) and MBP-Cep57 (1–242 amino acids) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by WB with anti-GST antibody and Coomassie blue staining. ( k ) Quantification of kinetochore signals of Cep57 and Mis12 in HeLa cells depleted of Cep57, Mis12 or negative control (NC) by siRNAs. The signal from control siRNA-treated cells was normalized to 1.0. More than 200 kinetochores from 20 cells were measured. The experiment was repeated three times. Data are mean±s.e.m. **** P

Techniques Used: Microscopy, Immunofluorescence, Staining, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Purification, Incubation, In Vitro, Magnetic Beads, Negative Control

Microtubule-binding activity of Cep57 contributes to checkpoint silencing. ( a ) Microtubule-binding assays in vitro . GST-Cep57 (195–500 amino acids; 0.1 μM) expressed in E. coli and Flag-Mad1 (0.05 μM) expressed in HEK293T cells were purified and incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. After centrifugation, supernatant (S) and pellet (P) were separated and used for Coomassie blue staining (top), and western blotting with anti-Flag antibody (bottom). ( b ) GST-Cep57 (195–500 amino acids; 0.1 μM)-coupled Glutathione Sepharose 4B beads were incubated with taxol-stabilized microtubules and purified Flag-Mad1 (0.05 μM) in BRB80 buffer at room temperature. The bead-bound proteins were analysed by western blotting with anti-Flag and anti-tubulin antibodies. GST-Cep57 (195–500 amino acids) was detected by Coomassie blue staining. ( c ) Microtubule-binding assays in vitro . Flag-Cep57 (0.05 μM) and Flag-Cep57-12A (0.05 μM) expressed in HEK293T cells and purified, and were incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. Samples were separated by centrifugation, and analysed by western blotting with anti-Flag antibody (top) and Coomassie blue staining (bottom). 12A: K432A, K434A, K435A, K438A, K441A, K442A, K467A, R469A, K473A, R474A, R475A and K476A. ( d ) Immunostaining of α-tubulin (red) in HeLa cells expressing Cep57-GFP or Cep57-12A-GFP. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. ( e ) Immunostaining of Flag-Cep57 (green) and Mad1 (red) in metaphase HeLa cells expressing RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A after transfection with Cep57-siRNA. DNA was stained with DAPI (blue). Scale bars, 5 μm. ( f ) Quantification of the percentage of metaphase cells with Mad1 signals at kinetochores from ( e ). Fifty cells were measured. ( g ) Quantification of the percentage of kinetochores with Mad1 signals in metaphase cells from ( e ). Greater than 100 kinetochores from 10 cells were measured. ( h ) Quantification of the percentage of metaphase cells in negative control (NC) or Cep57-depleted prometaphase and metaphase HeLa cells that expressed RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A. Mitotic stages were quantified by the morphology of DNA and spindles. Greater than 100 cells were measured. For f , g and h , the experiment was repeated three times. Data are mean±s.e.m. ** P
Figure Legend Snippet: Microtubule-binding activity of Cep57 contributes to checkpoint silencing. ( a ) Microtubule-binding assays in vitro . GST-Cep57 (195–500 amino acids; 0.1 μM) expressed in E. coli and Flag-Mad1 (0.05 μM) expressed in HEK293T cells were purified and incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. After centrifugation, supernatant (S) and pellet (P) were separated and used for Coomassie blue staining (top), and western blotting with anti-Flag antibody (bottom). ( b ) GST-Cep57 (195–500 amino acids; 0.1 μM)-coupled Glutathione Sepharose 4B beads were incubated with taxol-stabilized microtubules and purified Flag-Mad1 (0.05 μM) in BRB80 buffer at room temperature. The bead-bound proteins were analysed by western blotting with anti-Flag and anti-tubulin antibodies. GST-Cep57 (195–500 amino acids) was detected by Coomassie blue staining. ( c ) Microtubule-binding assays in vitro . Flag-Cep57 (0.05 μM) and Flag-Cep57-12A (0.05 μM) expressed in HEK293T cells and purified, and were incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. Samples were separated by centrifugation, and analysed by western blotting with anti-Flag antibody (top) and Coomassie blue staining (bottom). 12A: K432A, K434A, K435A, K438A, K441A, K442A, K467A, R469A, K473A, R474A, R475A and K476A. ( d ) Immunostaining of α-tubulin (red) in HeLa cells expressing Cep57-GFP or Cep57-12A-GFP. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. ( e ) Immunostaining of Flag-Cep57 (green) and Mad1 (red) in metaphase HeLa cells expressing RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A after transfection with Cep57-siRNA. DNA was stained with DAPI (blue). Scale bars, 5 μm. ( f ) Quantification of the percentage of metaphase cells with Mad1 signals at kinetochores from ( e ). Fifty cells were measured. ( g ) Quantification of the percentage of kinetochores with Mad1 signals in metaphase cells from ( e ). Greater than 100 kinetochores from 10 cells were measured. ( h ) Quantification of the percentage of metaphase cells in negative control (NC) or Cep57-depleted prometaphase and metaphase HeLa cells that expressed RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A. Mitotic stages were quantified by the morphology of DNA and spindles. Greater than 100 cells were measured. For f , g and h , the experiment was repeated three times. Data are mean±s.e.m. ** P

Techniques Used: Binding Assay, Activity Assay, In Vitro, Purification, Incubation, Centrifugation, Staining, Western Blot, Immunostaining, Expressing, Transfection, Negative Control

3) Product Images from "Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1–Mad2"

Article Title: Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1–Mad2

Journal: Nature Communications

doi: 10.1038/ncomms10151

Cep57 interacts with Mad1. ( a ) Mitotic HeLa cells arrested by nocodazole were used for immunoprecipitation (IP) with anti-Cep57 antibody and western blotting with anti-Cep57 and anti-Mad1 antibodies. IgG served as the negative control. ( b ) HEK293T cells were co-transfected with the indicated plasmids, and were used to for IP and western blotting. ( c ) Binding assays of Mad1 and Cep57. Flag-Cep57 and Mad1-GFP (expressed in HEK293T cells and purified) were incubated for IP with anti-GFP antibody. IP samples were analysed by western blotting with anti-Flag and anti-GFP antibodies. ( d ) In vitro pull-down assays of Mad1 and Cep57. GST-Mad1 (176–718 amino acids) and MBP-Cep57 (151–500 amino acids) (expressed in E. coli and purified) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by western blotting with anti-GST antibody and Coomassie blue staining. ( e ) GST pull-down assays of Cep57 and Mad1. Lysates of HEK293T cells overexpressing Mad1-GFP were incubated with Glutathione Sepharose 4B beads coated with GST, GST-Cep57 (1–242 amino acids) or GST-Cep57 (195–500 amino acids). The samples were analysed by western blotting with anti-GFP antibody. GST and GST-tagged proteins were stained with Coomassie blue. ( f ) Binding assays of Mad1 and Cep57 (195–500 amino acids). GST-Cep57 (195–500 amino acids; expressed in E. coli ) and Flag-Mad1 (expressed in HEK293T cells) were purified and incubated with anti-Flag antibody. The IP samples with anti-Flag antibody were analysed by western blotting with anti-GST and anti-Flag antibodies. ( g ) Schematic of truncated mutants of Mad1. ( h , i ) IP using lysates of HEK293T cells co-overexpressing GFP-Cep57 and Flag-Mad1 (FL, 1–530 and 531–718 amino acids) ( h ) or Flag-Mad1 (1–530, 1–175, 176–350 and 351–530 amino acids) ( i ) with anti-Flag antibody. The IP samples were analysed by western blotting with anti-GFP and anti-Flag antibodies. FL, full length. ( j ) Quantification and normalization of the kinetochore signal of Flag-Mad1 in HeLa cells that were transfected with FL and truncated mutants of Flag-Mad1 and treated with nocodazole for 1 h. Greater than 50 kinetochores from 5 cells were measured. The experiment was repeated three times. Data are mean±s.e.m.
Figure Legend Snippet: Cep57 interacts with Mad1. ( a ) Mitotic HeLa cells arrested by nocodazole were used for immunoprecipitation (IP) with anti-Cep57 antibody and western blotting with anti-Cep57 and anti-Mad1 antibodies. IgG served as the negative control. ( b ) HEK293T cells were co-transfected with the indicated plasmids, and were used to for IP and western blotting. ( c ) Binding assays of Mad1 and Cep57. Flag-Cep57 and Mad1-GFP (expressed in HEK293T cells and purified) were incubated for IP with anti-GFP antibody. IP samples were analysed by western blotting with anti-Flag and anti-GFP antibodies. ( d ) In vitro pull-down assays of Mad1 and Cep57. GST-Mad1 (176–718 amino acids) and MBP-Cep57 (151–500 amino acids) (expressed in E. coli and purified) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by western blotting with anti-GST antibody and Coomassie blue staining. ( e ) GST pull-down assays of Cep57 and Mad1. Lysates of HEK293T cells overexpressing Mad1-GFP were incubated with Glutathione Sepharose 4B beads coated with GST, GST-Cep57 (1–242 amino acids) or GST-Cep57 (195–500 amino acids). The samples were analysed by western blotting with anti-GFP antibody. GST and GST-tagged proteins were stained with Coomassie blue. ( f ) Binding assays of Mad1 and Cep57 (195–500 amino acids). GST-Cep57 (195–500 amino acids; expressed in E. coli ) and Flag-Mad1 (expressed in HEK293T cells) were purified and incubated with anti-Flag antibody. The IP samples with anti-Flag antibody were analysed by western blotting with anti-GST and anti-Flag antibodies. ( g ) Schematic of truncated mutants of Mad1. ( h , i ) IP using lysates of HEK293T cells co-overexpressing GFP-Cep57 and Flag-Mad1 (FL, 1–530 and 531–718 amino acids) ( h ) or Flag-Mad1 (1–530, 1–175, 176–350 and 351–530 amino acids) ( i ) with anti-Flag antibody. The IP samples were analysed by western blotting with anti-GFP and anti-Flag antibodies. FL, full length. ( j ) Quantification and normalization of the kinetochore signal of Flag-Mad1 in HeLa cells that were transfected with FL and truncated mutants of Flag-Mad1 and treated with nocodazole for 1 h. Greater than 50 kinetochores from 5 cells were measured. The experiment was repeated three times. Data are mean±s.e.m.

Techniques Used: Immunoprecipitation, Western Blot, Negative Control, Transfection, Binding Assay, Purification, Incubation, In Vitro, Magnetic Beads, Staining

Cep57 is a novel kinetochore component in human cells. ( a ) Three-dimensional structured illumination microscopy (SIM) images of HeLa cells double-immunostained with antibodies against Cep57 (green) and CREST (red). ( b ) Immunofluorescence of Cep57 (green) and CENP-A (red) in HeLa cells. ( c ) Immunofluorescence of Cep57 (green) and Mis12 (red) in HeLa cells at metaphase after treatment with MG132 for 1 h. A linescan through the kinetochore pair indicates the co-localization of Cep57 with Mis12. ( d ) Stimulated emission depletion (STED) images of immunofluorescence of Cep57 (green) and Zwint-1 (red) in RPE1 cells. ( e ) Immunofluorescence of Cep57 (green) and CREST (red) in HeLa cells at different stages during mitosis. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). ( f ) HEK293T cells were co-transfected with Cep57-GFP and Mis12-HA. The cell lysates were immunoprecipitated (IP) and analysed by western blotting (WB) with the indicated antibodies. ( g ) Binding assays of Mis12 and Cep57. GST-Mis12 (expressed in E. coli and purified) was incubated with Flag-Cep57 (expressed in HEK293T cells and purified). The IP samples with anti-Flag antibody were analysed by WB with anti-Flag and anti-Mis12 antibodies. ( h ) Schematic of Cep57 truncated mutants. ( i ) GST pull-down assays of Cep57 and Mis12. Lysates of HEK293T cells overexpressing Mis12-HA were incubated with Glutathione Sepharose 4B beads coated with GST or GST-Cep57N/C. The samples were analysed by WB with anti-HA antibody. GST-tagged proteins were stained with Coomassie blue. ( j ) In vitro pull-down assays of Mis12 and Cep57 (1–242 amino acids). GST-Mis12 (expressed in E. coli and purified) and MBP-Cep57 (1–242 amino acids) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by WB with anti-GST antibody and Coomassie blue staining. ( k ) Quantification of kinetochore signals of Cep57 and Mis12 in HeLa cells depleted of Cep57, Mis12 or negative control (NC) by siRNAs. The signal from control siRNA-treated cells was normalized to 1.0. More than 200 kinetochores from 20 cells were measured. The experiment was repeated three times. Data are mean±s.e.m. **** P
Figure Legend Snippet: Cep57 is a novel kinetochore component in human cells. ( a ) Three-dimensional structured illumination microscopy (SIM) images of HeLa cells double-immunostained with antibodies against Cep57 (green) and CREST (red). ( b ) Immunofluorescence of Cep57 (green) and CENP-A (red) in HeLa cells. ( c ) Immunofluorescence of Cep57 (green) and Mis12 (red) in HeLa cells at metaphase after treatment with MG132 for 1 h. A linescan through the kinetochore pair indicates the co-localization of Cep57 with Mis12. ( d ) Stimulated emission depletion (STED) images of immunofluorescence of Cep57 (green) and Zwint-1 (red) in RPE1 cells. ( e ) Immunofluorescence of Cep57 (green) and CREST (red) in HeLa cells at different stages during mitosis. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). ( f ) HEK293T cells were co-transfected with Cep57-GFP and Mis12-HA. The cell lysates were immunoprecipitated (IP) and analysed by western blotting (WB) with the indicated antibodies. ( g ) Binding assays of Mis12 and Cep57. GST-Mis12 (expressed in E. coli and purified) was incubated with Flag-Cep57 (expressed in HEK293T cells and purified). The IP samples with anti-Flag antibody were analysed by WB with anti-Flag and anti-Mis12 antibodies. ( h ) Schematic of Cep57 truncated mutants. ( i ) GST pull-down assays of Cep57 and Mis12. Lysates of HEK293T cells overexpressing Mis12-HA were incubated with Glutathione Sepharose 4B beads coated with GST or GST-Cep57N/C. The samples were analysed by WB with anti-HA antibody. GST-tagged proteins were stained with Coomassie blue. ( j ) In vitro pull-down assays of Mis12 and Cep57 (1–242 amino acids). GST-Mis12 (expressed in E. coli and purified) and MBP-Cep57 (1–242 amino acids) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by WB with anti-GST antibody and Coomassie blue staining. ( k ) Quantification of kinetochore signals of Cep57 and Mis12 in HeLa cells depleted of Cep57, Mis12 or negative control (NC) by siRNAs. The signal from control siRNA-treated cells was normalized to 1.0. More than 200 kinetochores from 20 cells were measured. The experiment was repeated three times. Data are mean±s.e.m. **** P

Techniques Used: Microscopy, Immunofluorescence, Staining, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Purification, Incubation, In Vitro, Magnetic Beads, Negative Control

Microtubule-binding activity of Cep57 contributes to checkpoint silencing. ( a ) Microtubule-binding assays in vitro . GST-Cep57 (195–500 amino acids; 0.1 μM) expressed in E. coli and Flag-Mad1 (0.05 μM) expressed in HEK293T cells were purified and incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. After centrifugation, supernatant (S) and pellet (P) were separated and used for Coomassie blue staining (top), and western blotting with anti-Flag antibody (bottom). ( b ) GST-Cep57 (195–500 amino acids; 0.1 μM)-coupled Glutathione Sepharose 4B beads were incubated with taxol-stabilized microtubules and purified Flag-Mad1 (0.05 μM) in BRB80 buffer at room temperature. The bead-bound proteins were analysed by western blotting with anti-Flag and anti-tubulin antibodies. GST-Cep57 (195–500 amino acids) was detected by Coomassie blue staining. ( c ) Microtubule-binding assays in vitro . Flag-Cep57 (0.05 μM) and Flag-Cep57-12A (0.05 μM) expressed in HEK293T cells and purified, and were incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. Samples were separated by centrifugation, and analysed by western blotting with anti-Flag antibody (top) and Coomassie blue staining (bottom). 12A: K432A, K434A, K435A, K438A, K441A, K442A, K467A, R469A, K473A, R474A, R475A and K476A. ( d ) Immunostaining of α-tubulin (red) in HeLa cells expressing Cep57-GFP or Cep57-12A-GFP. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. ( e ) Immunostaining of Flag-Cep57 (green) and Mad1 (red) in metaphase HeLa cells expressing RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A after transfection with Cep57-siRNA. DNA was stained with DAPI (blue). Scale bars, 5 μm. ( f ) Quantification of the percentage of metaphase cells with Mad1 signals at kinetochores from ( e ). Fifty cells were measured. ( g ) Quantification of the percentage of kinetochores with Mad1 signals in metaphase cells from ( e ). Greater than 100 kinetochores from 10 cells were measured. ( h ) Quantification of the percentage of metaphase cells in negative control (NC) or Cep57-depleted prometaphase and metaphase HeLa cells that expressed RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A. Mitotic stages were quantified by the morphology of DNA and spindles. Greater than 100 cells were measured. For f , g and h , the experiment was repeated three times. Data are mean±s.e.m. ** P
Figure Legend Snippet: Microtubule-binding activity of Cep57 contributes to checkpoint silencing. ( a ) Microtubule-binding assays in vitro . GST-Cep57 (195–500 amino acids; 0.1 μM) expressed in E. coli and Flag-Mad1 (0.05 μM) expressed in HEK293T cells were purified and incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. After centrifugation, supernatant (S) and pellet (P) were separated and used for Coomassie blue staining (top), and western blotting with anti-Flag antibody (bottom). ( b ) GST-Cep57 (195–500 amino acids; 0.1 μM)-coupled Glutathione Sepharose 4B beads were incubated with taxol-stabilized microtubules and purified Flag-Mad1 (0.05 μM) in BRB80 buffer at room temperature. The bead-bound proteins were analysed by western blotting with anti-Flag and anti-tubulin antibodies. GST-Cep57 (195–500 amino acids) was detected by Coomassie blue staining. ( c ) Microtubule-binding assays in vitro . Flag-Cep57 (0.05 μM) and Flag-Cep57-12A (0.05 μM) expressed in HEK293T cells and purified, and were incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. Samples were separated by centrifugation, and analysed by western blotting with anti-Flag antibody (top) and Coomassie blue staining (bottom). 12A: K432A, K434A, K435A, K438A, K441A, K442A, K467A, R469A, K473A, R474A, R475A and K476A. ( d ) Immunostaining of α-tubulin (red) in HeLa cells expressing Cep57-GFP or Cep57-12A-GFP. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. ( e ) Immunostaining of Flag-Cep57 (green) and Mad1 (red) in metaphase HeLa cells expressing RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A after transfection with Cep57-siRNA. DNA was stained with DAPI (blue). Scale bars, 5 μm. ( f ) Quantification of the percentage of metaphase cells with Mad1 signals at kinetochores from ( e ). Fifty cells were measured. ( g ) Quantification of the percentage of kinetochores with Mad1 signals in metaphase cells from ( e ). Greater than 100 kinetochores from 10 cells were measured. ( h ) Quantification of the percentage of metaphase cells in negative control (NC) or Cep57-depleted prometaphase and metaphase HeLa cells that expressed RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A. Mitotic stages were quantified by the morphology of DNA and spindles. Greater than 100 cells were measured. For f , g and h , the experiment was repeated three times. Data are mean±s.e.m. ** P

Techniques Used: Binding Assay, Activity Assay, In Vitro, Purification, Incubation, Centrifugation, Staining, Western Blot, Immunostaining, Expressing, Transfection, Negative Control

4) Product Images from "Dysferlin Interacts with Tubulin and Microtubules in Mouse Skeletal Muscle"

Article Title: Dysferlin Interacts with Tubulin and Microtubules in Mouse Skeletal Muscle

Journal: PLoS ONE

doi: 10.1371/journal.pone.0010122

Dysferlin complexes with alpha-tubulin. A. GST, GST-TubA4A and GST-TubA1B fusion proteins immobilized onto glutathione-Sepharose 4B beads were incubated with mouse skeletal muscle homogenate. GST, GST-TubA4A or GST-TubA1B with adsorbed proteins from mouse skeletal muscle were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with mouse monoclonal anti-dysferlin antibody. Left panel: nitrocellulose membrane stained with ponceau red, right panel: detection of immunoreactive dysferlin. B. GFP-dysferlin was overexpressed in HEK293T cells and then immunoprecipitated from cell extracts with anti-GFP antibody. As a control, protein A-Sepharose beads coated with anti-GFP antibody were incubated with extracts of non-transfected HEK293T cells. Proteins were separated on SDS-PAGE gel and were transferred onto a nitrocellulose membrane and blotted with anti-GFP or anti-alpha-tubulin antibodies. Input (right panel), immunoprecipitate (left panel). C–D. Co-immunoprecipitation of dysferlin with anti-alpha-tubulin antibody from C2C12 myotube extracts ( C ) or mouse skeletal muscle homogenate ( D ). Input (right panel), immunoprecipitate (left panel). As a control (CTL), protein A-Sepharose beads were incubated with myotube extracts in the absence of anti-alpha-tubulin antibody.
Figure Legend Snippet: Dysferlin complexes with alpha-tubulin. A. GST, GST-TubA4A and GST-TubA1B fusion proteins immobilized onto glutathione-Sepharose 4B beads were incubated with mouse skeletal muscle homogenate. GST, GST-TubA4A or GST-TubA1B with adsorbed proteins from mouse skeletal muscle were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with mouse monoclonal anti-dysferlin antibody. Left panel: nitrocellulose membrane stained with ponceau red, right panel: detection of immunoreactive dysferlin. B. GFP-dysferlin was overexpressed in HEK293T cells and then immunoprecipitated from cell extracts with anti-GFP antibody. As a control, protein A-Sepharose beads coated with anti-GFP antibody were incubated with extracts of non-transfected HEK293T cells. Proteins were separated on SDS-PAGE gel and were transferred onto a nitrocellulose membrane and blotted with anti-GFP or anti-alpha-tubulin antibodies. Input (right panel), immunoprecipitate (left panel). C–D. Co-immunoprecipitation of dysferlin with anti-alpha-tubulin antibody from C2C12 myotube extracts ( C ) or mouse skeletal muscle homogenate ( D ). Input (right panel), immunoprecipitate (left panel). As a control (CTL), protein A-Sepharose beads were incubated with myotube extracts in the absence of anti-alpha-tubulin antibody.

Techniques Used: Incubation, SDS Page, Staining, Immunoprecipitation, Transfection, CTL Assay

Alpha-tubulin interacts with dysferlin in a calcium-independent manner. A. Upper panel: Myoblast cell extracts were incubated with GST alone or the various GST-dysferlin C2 domain fusion proteins precoupled to glutathione-Sepharose 4B beads in the absence (−) or presence (+) of 1 mM calcium. The bound proteins were separated on SDS-PAGE followed by Western blot analysis using anti-alpha-tubulin antibody. Lower panel: nitrocellulose membrane of GST-dysferlin C2 domains with adsorbed proteins from the cell extract stained with ponceau red. B. Co-immunoprecipitation of alpha-tubulin and dysferlin with anti-alpha-tubulin antibody from mouse skeletal muscle homogenate in the presence of increasing calcium concentrations. Proteins were separated and detected with anti-alpha-tubulin and anti-dysferlin antibodies. As a control (CTL), protein A-Sepharose beads were incubated with muscle homogenate in the absence of anti-alpha-tubulin antibody.
Figure Legend Snippet: Alpha-tubulin interacts with dysferlin in a calcium-independent manner. A. Upper panel: Myoblast cell extracts were incubated with GST alone or the various GST-dysferlin C2 domain fusion proteins precoupled to glutathione-Sepharose 4B beads in the absence (−) or presence (+) of 1 mM calcium. The bound proteins were separated on SDS-PAGE followed by Western blot analysis using anti-alpha-tubulin antibody. Lower panel: nitrocellulose membrane of GST-dysferlin C2 domains with adsorbed proteins from the cell extract stained with ponceau red. B. Co-immunoprecipitation of alpha-tubulin and dysferlin with anti-alpha-tubulin antibody from mouse skeletal muscle homogenate in the presence of increasing calcium concentrations. Proteins were separated and detected with anti-alpha-tubulin and anti-dysferlin antibodies. As a control (CTL), protein A-Sepharose beads were incubated with muscle homogenate in the absence of anti-alpha-tubulin antibody.

Techniques Used: Incubation, SDS Page, Western Blot, Staining, Immunoprecipitation, CTL Assay

Alpha-tubulin interacts with dysferlin through the C2A and C2B domains. A. Schematic illustration of full-length wild type dysferlin and the various GST-C2 domain constructs used. B. C2C12 myoblast extract was incubated with GST alone or with the various GST-C2 domains precoupled to glutathione-Sepharose 4B beads. The bound proteins were separated on SDS-PAGE followed by Western blot analysis using anti-alpha-tubulin antibody. SM: standard material. Lower panel: nitrocellulose membrane of GST-dysferlin C2 domains with adsorbed proteins from the cell extract stained with ponceau red.
Figure Legend Snippet: Alpha-tubulin interacts with dysferlin through the C2A and C2B domains. A. Schematic illustration of full-length wild type dysferlin and the various GST-C2 domain constructs used. B. C2C12 myoblast extract was incubated with GST alone or with the various GST-C2 domains precoupled to glutathione-Sepharose 4B beads. The bound proteins were separated on SDS-PAGE followed by Western blot analysis using anti-alpha-tubulin antibody. SM: standard material. Lower panel: nitrocellulose membrane of GST-dysferlin C2 domains with adsorbed proteins from the cell extract stained with ponceau red.

Techniques Used: Construct, Incubation, SDS Page, Western Blot, Staining

5) Product Images from "A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6"

Article Title: A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064149

Detection of the Mint1 826 protein. For detection of the Mint1 826 protein GST pulldown assays were performed. 10 µl of Glutathione Sepharose™ 4B beads were coated with 10 µg of GST or the denoted GST fusion protein and then incubated with 1 mg of mouse brain lysate for 3 h at 4°C. Since Mint1 826 seems to exhibit a relatively low expression level, three samples were pooled for Western blot analyses. input: 15 µg mouse brain lysate.
Figure Legend Snippet: Detection of the Mint1 826 protein. For detection of the Mint1 826 protein GST pulldown assays were performed. 10 µl of Glutathione Sepharose™ 4B beads were coated with 10 µg of GST or the denoted GST fusion protein and then incubated with 1 mg of mouse brain lysate for 3 h at 4°C. Since Mint1 826 seems to exhibit a relatively low expression level, three samples were pooled for Western blot analyses. input: 15 µg mouse brain lysate.

Techniques Used: Incubation, Expressing, Western Blot

Verification of the interaction between Mint1 826 and APP. 10 µl of Glutathione Sepharose™ 4B beads were coated with 5 µg of GST or the denoted GST Mint1 fusion proteins and then incubated with MEF dko APP695 AA12 cell lysates (20 x input) for 1.5 h at 4°C. Samples were analyzed by Western blotting using an anti-APP C-terminus antibody.
Figure Legend Snippet: Verification of the interaction between Mint1 826 and APP. 10 µl of Glutathione Sepharose™ 4B beads were coated with 5 µg of GST or the denoted GST Mint1 fusion proteins and then incubated with MEF dko APP695 AA12 cell lysates (20 x input) for 1.5 h at 4°C. Samples were analyzed by Western blotting using an anti-APP C-terminus antibody.

Techniques Used: Incubation, Western Blot

Verification of the interaction between Mint1 826 and Rab6 isoforms using GST pulldown experiments. 10 µl of Glutathione Sepharose™ 4B beads were coated with GST or the denoted GST fusion protein and then incubated with the designated prey protein. Samples were analyzed by Western blotting using an anti-GFP (a) or anti-Mint1 (b) antibody. QL: constitutively active variant (Q72L), TN: inactive variant (T27N). A) 5 µg of the GST Mint 1 fusion proteins were incubated with lysates of stably transfected HeLa T-REx™ cells overexpressing Rab6 GFP or CFP fusion proteins (20×input) for 1.5 h at 4°C. B) 1 µg of the GST Rab6 fusion protein was incubated with 300 ng of Mint 826 for 1 h at 4°C. Mint1 826 was isolated from thrombin cleaved bacterially expressed GST Mint1 826. input: 50 ng Mint1 826.
Figure Legend Snippet: Verification of the interaction between Mint1 826 and Rab6 isoforms using GST pulldown experiments. 10 µl of Glutathione Sepharose™ 4B beads were coated with GST or the denoted GST fusion protein and then incubated with the designated prey protein. Samples were analyzed by Western blotting using an anti-GFP (a) or anti-Mint1 (b) antibody. QL: constitutively active variant (Q72L), TN: inactive variant (T27N). A) 5 µg of the GST Mint 1 fusion proteins were incubated with lysates of stably transfected HeLa T-REx™ cells overexpressing Rab6 GFP or CFP fusion proteins (20×input) for 1.5 h at 4°C. B) 1 µg of the GST Rab6 fusion protein was incubated with 300 ng of Mint 826 for 1 h at 4°C. Mint1 826 was isolated from thrombin cleaved bacterially expressed GST Mint1 826. input: 50 ng Mint1 826.

Techniques Used: Incubation, Western Blot, Variant Assay, Stable Transfection, Transfection, Isolation

6) Product Images from "Functional expression of the epithelial Ca2+ channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex"

Article Title: Functional expression of the epithelial Ca2+ channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg162

Fig. 1. Interaction of TRPV5 and S100A10 as shown by yeast two- hybrid and GST pull-down analyses. ( A ) The C-terminal tail of TRPV5 or γENaC and full-length S100A10 were cotransformed into the Y153 yeast strain and grown on media without tryptophan and leucine. ( B ) β-galactosidase activity was demonstrated in TRPV5 and S100A10 cotransformed yeast, whereas no activity was observed in γENaC and S100A10 cotransformed yeast. Two representative colonies are depicted. ( C ) Lysates of X.laevis oocytes injected with 20 ng of VSV-tagged S100A10 cRNA were incubated with GST or GST fused to the C-terminal tail of TRPV5 or TRPV6 immobilized on glutathione– Sepharose 4B beads. S100A10 interacted specifically with TRPV5 and TRPV6, but not with GST alone. ( D ) The experiment was performed as outlined in (C). Binding of S100A10 to TRPV5 was demonstrated in the presence of 1 mM Ca 2+ or 2 mM EDTA. ( E ) [ 35 S]methionine- labeled full-length TRPV5 or TRPV6 was incubated with GST or GST–S100A10 immobilized on glutathione–Sepharose 4B beads. Both TRPV5 and TRPV6 interacted with S100A10, whereas no binding to GST alone was observed.
Figure Legend Snippet: Fig. 1. Interaction of TRPV5 and S100A10 as shown by yeast two- hybrid and GST pull-down analyses. ( A ) The C-terminal tail of TRPV5 or γENaC and full-length S100A10 were cotransformed into the Y153 yeast strain and grown on media without tryptophan and leucine. ( B ) β-galactosidase activity was demonstrated in TRPV5 and S100A10 cotransformed yeast, whereas no activity was observed in γENaC and S100A10 cotransformed yeast. Two representative colonies are depicted. ( C ) Lysates of X.laevis oocytes injected with 20 ng of VSV-tagged S100A10 cRNA were incubated with GST or GST fused to the C-terminal tail of TRPV5 or TRPV6 immobilized on glutathione– Sepharose 4B beads. S100A10 interacted specifically with TRPV5 and TRPV6, but not with GST alone. ( D ) The experiment was performed as outlined in (C). Binding of S100A10 to TRPV5 was demonstrated in the presence of 1 mM Ca 2+ or 2 mM EDTA. ( E ) [ 35 S]methionine- labeled full-length TRPV5 or TRPV6 was incubated with GST or GST–S100A10 immobilized on glutathione–Sepharose 4B beads. Both TRPV5 and TRPV6 interacted with S100A10, whereas no binding to GST alone was observed.

Techniques Used: Activity Assay, Injection, Incubation, Binding Assay, Labeling

Fig. 4. Mapping of the S100A10 binding site in TRPV5. GST fusion proteins containing different portions of the C-terminal tail of TRPV5 were constructed according to the schematic drawing. These proteins were immobilized on glutathione–Sepharose 4B beads and then incu bated with lysates from X.laevis oocytes injected with 20 ng of S100A10 cRNA. Interaction of S100A10 with the GST fusion proteins was determined by immunoblotting. The binding site was localized between amino acids 598 and 603. Virtually all interaction with S100A10 was abolished when this region (VATTV) was mutated into glycines. A similar effect was observed with the single point mutant TRPV5 T600A.
Figure Legend Snippet: Fig. 4. Mapping of the S100A10 binding site in TRPV5. GST fusion proteins containing different portions of the C-terminal tail of TRPV5 were constructed according to the schematic drawing. These proteins were immobilized on glutathione–Sepharose 4B beads and then incu bated with lysates from X.laevis oocytes injected with 20 ng of S100A10 cRNA. Interaction of S100A10 with the GST fusion proteins was determined by immunoblotting. The binding site was localized between amino acids 598 and 603. Virtually all interaction with S100A10 was abolished when this region (VATTV) was mutated into glycines. A similar effect was observed with the single point mutant TRPV5 T600A.

Techniques Used: Binding Assay, Construct, Injection, Mutagenesis

Fig. 2. Annexin 2 interacts with TRPV5 via S100A10. Xenopus laevis oocytes were injected with S100A10 or co-injected with annexin 2 and VSV-tagged S100A10 cRNAs. ( A ) Lysates of S100A10 cRNA-injected oocytes were treated with DTBP and analyzed by immunoblot. The chemically cross-linked S100A10 band runs at 23 kDa, exactly the expected size of a VSV-tagged S100A10 dimer. Homogenates of non-injected and oocytes co-injected with S100A10 and annexin 2 cRNAs were subjected to immunoprecipitation using monoclonal anti-VSV antibodies. Annexin 2 co-immunoprecipitated with S100A10 as was visualized by autoradiography of the metabolically labeled proteins. As a control, the expression of S100A10 and annexin 2 in the co-injected oocytes was demonstrated by immunoblot analysis to demonstrate that the precipitated proteins were of the correct size. ( B ) Homogenates of annexin 2 cRNA-injected or S100A10 and annexin 2 cRNA-co-injected oocytes were incubated with GST alone or GST fusion protein containing the TRPV5 C-terminal tail immobilized on glutathione–Sepharose 4B beads. The association of annexin 2 with TRPV5 in the presence of S100A10 was demonstrated by immunoblot using a monoclonal anti-annexin 2 antibody. ( C ) Full-length annexin 2, S100A10 and TRPV5 were in vitro translated using a reticulocyte lysate system in the presence of canine microsomal membranes. S100A10 and annexin 2 were co-immunoprecipitated with TRPV5 confirming the formation of a TRPV5–S100A10–annexin 2 complex.
Figure Legend Snippet: Fig. 2. Annexin 2 interacts with TRPV5 via S100A10. Xenopus laevis oocytes were injected with S100A10 or co-injected with annexin 2 and VSV-tagged S100A10 cRNAs. ( A ) Lysates of S100A10 cRNA-injected oocytes were treated with DTBP and analyzed by immunoblot. The chemically cross-linked S100A10 band runs at 23 kDa, exactly the expected size of a VSV-tagged S100A10 dimer. Homogenates of non-injected and oocytes co-injected with S100A10 and annexin 2 cRNAs were subjected to immunoprecipitation using monoclonal anti-VSV antibodies. Annexin 2 co-immunoprecipitated with S100A10 as was visualized by autoradiography of the metabolically labeled proteins. As a control, the expression of S100A10 and annexin 2 in the co-injected oocytes was demonstrated by immunoblot analysis to demonstrate that the precipitated proteins were of the correct size. ( B ) Homogenates of annexin 2 cRNA-injected or S100A10 and annexin 2 cRNA-co-injected oocytes were incubated with GST alone or GST fusion protein containing the TRPV5 C-terminal tail immobilized on glutathione–Sepharose 4B beads. The association of annexin 2 with TRPV5 in the presence of S100A10 was demonstrated by immunoblot using a monoclonal anti-annexin 2 antibody. ( C ) Full-length annexin 2, S100A10 and TRPV5 were in vitro translated using a reticulocyte lysate system in the presence of canine microsomal membranes. S100A10 and annexin 2 were co-immunoprecipitated with TRPV5 confirming the formation of a TRPV5–S100A10–annexin 2 complex.

Techniques Used: Injection, Immunoprecipitation, Autoradiography, Metabolic Labelling, Labeling, Expressing, Incubation, In Vitro

7) Product Images from "Canonical SecA Associates with an Accessory Secretory Protein Complex Involved in Biogenesis of a Streptococcal Serine-Rich Repeat Glycoprotein ▿"

Article Title: Canonical SecA Associates with an Accessory Secretory Protein Complex Involved in Biogenesis of a Streptococcal Serine-Rich Repeat Glycoprotein ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.05668-11

In vitro GST pulldown assays for SecA2, Asp1, and Asp3 interactions by S. agalactiae . (A) Equal amounts of purified GST, GST-Asp3, and GST-SecA2 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-Asp1. (B) Equal amounts of purified GST, GST-Asp1, and GST-SecA2 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-Asp3. (C) Equal amounts of purified GST, GST-Asp1, and GST-Asp3 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-SecA2. The pull-down protein complexes were analyzed by Western blotting using anti-c-Myc monoclonal antibody. Inputs represent the in vitro -translated protein products.
Figure Legend Snippet: In vitro GST pulldown assays for SecA2, Asp1, and Asp3 interactions by S. agalactiae . (A) Equal amounts of purified GST, GST-Asp3, and GST-SecA2 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-Asp1. (B) Equal amounts of purified GST, GST-Asp1, and GST-SecA2 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-Asp3. (C) Equal amounts of purified GST, GST-Asp1, and GST-Asp3 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-SecA2. The pull-down protein complexes were analyzed by Western blotting using anti-c-Myc monoclonal antibody. Inputs represent the in vitro -translated protein products.

Techniques Used: In Vitro, Purification, Incubation, Western Blot

In vitro GST pulldown assays to detect SecA2, Gap1, and Gap3 interactions. (A) Equal amounts of purified GST and GST-SecA2 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-Gap1. (B) Equal amounts of purified GST, GST-Gap1, and GST-Gap3 bound to glutathione Sepharose 4B beads were incubated with in vitro -translated c-Myc-SecA2. The pulldown protein complexes were analyzed by Western blotting using c-Myc monoclonal antibody. Inputs are the in vitro -translated protein products.
Figure Legend Snippet: In vitro GST pulldown assays to detect SecA2, Gap1, and Gap3 interactions. (A) Equal amounts of purified GST and GST-SecA2 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-Gap1. (B) Equal amounts of purified GST, GST-Gap1, and GST-Gap3 bound to glutathione Sepharose 4B beads were incubated with in vitro -translated c-Myc-SecA2. The pulldown protein complexes were analyzed by Western blotting using c-Myc monoclonal antibody. Inputs are the in vitro -translated protein products.

Techniques Used: In Vitro, Purification, Incubation, Western Blot

In vitro GST pulldown assays for interactions between SecA and the SecA2-Gap1-Gap3 complex. (A) Equal amounts of purified GST, GST-Gap3, and GST-SecA bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-SecA2. (B) Equal amounts of purified GST and GST-SecA bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-Gap3. (C) Equal amounts of purified GST and GST-Gap3 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-SecA. The pulldown protein complexes were analyzed by Western blotting using anti-c-Myc monoclonal antibody. Inputs represent the in vitro -translated protein products.
Figure Legend Snippet: In vitro GST pulldown assays for interactions between SecA and the SecA2-Gap1-Gap3 complex. (A) Equal amounts of purified GST, GST-Gap3, and GST-SecA bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-SecA2. (B) Equal amounts of purified GST and GST-SecA bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-Gap3. (C) Equal amounts of purified GST and GST-Gap3 bound to glutathione-Sepharose 4B beads were incubated with in vitro -translated c-Myc-SecA. The pulldown protein complexes were analyzed by Western blotting using anti-c-Myc monoclonal antibody. Inputs represent the in vitro -translated protein products.

Techniques Used: In Vitro, Purification, Incubation, Western Blot

8) Product Images from "Mechanistic Insight Into the Interaction Between Helicobacter pylori Urease Subunit α and Its Molecular Chaperone Hsp60"

Article Title: Mechanistic Insight Into the Interaction Between Helicobacter pylori Urease Subunit α and Its Molecular Chaperone Hsp60

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.00153

Purification of recombinant proteins with GST tags for pull-down assays. (A) UreA fused with GST tag on its N-terminus (GST-UreA). (B) UreB fused with GST tag on its N-terminus (GST-UreB). (C) GST tag. (D) UreA fused with GST tag on its C-terminus (UreA-GST). (E) UreB fused with GST tag on its C-terminus (UreB-GST). The recombinant proteins with GST tags were all purified as follows: The bacterial cells were disrupted in Tris-HCl (50 mM, pH 8.0) and centrifuged at 11,000 rpm for 30 min. The supernatant was then incubated with glutathione Sepharose 4B beads overnight with gentle inversion. Next, the beads were washed three times with Tris-HCl (50 mM, pH 8.0) and eluted with glutathione (reduced form) to a final concentration of 10 mM. During the purification of UreA-GST and UreB-GST, a large amount of cleaved GST tag (about 26 kDa) was also eluted from the beads.
Figure Legend Snippet: Purification of recombinant proteins with GST tags for pull-down assays. (A) UreA fused with GST tag on its N-terminus (GST-UreA). (B) UreB fused with GST tag on its N-terminus (GST-UreB). (C) GST tag. (D) UreA fused with GST tag on its C-terminus (UreA-GST). (E) UreB fused with GST tag on its C-terminus (UreB-GST). The recombinant proteins with GST tags were all purified as follows: The bacterial cells were disrupted in Tris-HCl (50 mM, pH 8.0) and centrifuged at 11,000 rpm for 30 min. The supernatant was then incubated with glutathione Sepharose 4B beads overnight with gentle inversion. Next, the beads were washed three times with Tris-HCl (50 mM, pH 8.0) and eluted with glutathione (reduced form) to a final concentration of 10 mM. During the purification of UreA-GST and UreB-GST, a large amount of cleaved GST tag (about 26 kDa) was also eluted from the beads.

Techniques Used: Purification, Recombinant, Incubation, Concentration Assay

Pull-down assay. (A) Results of GST pull-down assay to detect the interaction of GST-UreA and GST-UreB with Hsp60. (B) The interaction of Hsp60 with UreA-GST or UreB-GST was verified individually. All pull-down assays were performed as follows: 20 μl of glutathione Sepharose 4B beads saturated with GST-fused proteins were incubated with 0.4 ml of Hsp60 (1 mg/ml dissolved in 50 mM Tris-HCl, pH 8.0) at 4°C for 2 h. The pellets were washed four times and resuspended in SDS-PAGE sample buffer for Western blotting (W) and SDS-PAGE (S) analysis. Hsp60 co-incubated with GST-tag immobilized beads and empty beads were used as negative controls. Purified Hsp60 was used as a positive control. Anti-His antibody was used to detect the bound Hsp60 and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG 158 was used to detect the primary antibody. All experiments were repeated at least three times.
Figure Legend Snippet: Pull-down assay. (A) Results of GST pull-down assay to detect the interaction of GST-UreA and GST-UreB with Hsp60. (B) The interaction of Hsp60 with UreA-GST or UreB-GST was verified individually. All pull-down assays were performed as follows: 20 μl of glutathione Sepharose 4B beads saturated with GST-fused proteins were incubated with 0.4 ml of Hsp60 (1 mg/ml dissolved in 50 mM Tris-HCl, pH 8.0) at 4°C for 2 h. The pellets were washed four times and resuspended in SDS-PAGE sample buffer for Western blotting (W) and SDS-PAGE (S) analysis. Hsp60 co-incubated with GST-tag immobilized beads and empty beads were used as negative controls. Purified Hsp60 was used as a positive control. Anti-His antibody was used to detect the bound Hsp60 and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG 158 was used to detect the primary antibody. All experiments were repeated at least three times.

Techniques Used: Pull Down Assay, Incubation, SDS Page, Western Blot, Purification, Positive Control

9) Product Images from "Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells"

Article Title: Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.339

Co-factor(s) for nuclear import of TG2. Recombinant human TG2 (about 1.5 pmol) was incubated for 1 h at room temperature with glutathione sepharose 4B beads conjugated with six times molar excess of ( a ) GST (lane 1), GST-tagged importin- β (lane 2), - α 5 (lane 3), - α 1 (lane 4) and - α 3 (lane 7) or with GST-tagged importin- α 5, α 1, α 3 in presence of HA-tagged importin- β in (lane 4, 6 and 8 respectively). ( b ) GST (lane 1) or GST-importins- α 3/HA-importin- β complex (lane 2) or in presence of peptide SV40 NLS, TG2 NES and TG2 NLS (lane 3, 4 and 5, respectively) ( c ) GST (lane 1) or GST-tagged exportin-1 (Exp-1) in the presence or absence of 0.1% EtOH, 1 mM ACR and 10 μ g/ml LMB as indicated. After spin-down, proteins were eluted with SDS-PAGE sample buffer and TG2 level in each pull down obtained under each condition were determined by western blotting using an antibody against TG2. A representative image, showing intensity value of each blot relative to lane 2 is presented as mean for three independent experiments ( a ) or as mean for two independent experiments ( b and c ). Specificity in the immunoprecipitation experiment of the TG2-exportin-1 complex was ensured in the control experiment ( Supplementary Figure 7 ). ( d ) JHH-7 cells were seeded at 1 × 10 3 cells per well in 96-well plate and incubated at 37 °C overnight. PLA was performed according to the manufacturer's instruction. In control (row 1), the cells were treated with media for 3 h and cells were fixed, permeabilized and no antibodies were used against TG2 and importins for PLA assay. While in others, cells were treated with 0.1% EtOH for 3 h, fixed, permeabilized and treated with combinations of mouse anti-TG2 (CUB7402) and rabbit anti-KPNA4 (left panel, row 2), mouse anti-TG2 (CUB7402) and rabbit anti-SRP-1 (central panel, row 2) or mouse anti-TG2 (CUB7402) and rabbit anti-importin- β (right panel, row 2). After amplification and staining with H33258, the cells were observed under a confocal microscope. Red fluorescence dots derived from amplification of detected protein interaction was monitored with blue fluorescence from H33258. A scale bar=20 μ m. A representative image from three independent experiments with similar results is presented
Figure Legend Snippet: Co-factor(s) for nuclear import of TG2. Recombinant human TG2 (about 1.5 pmol) was incubated for 1 h at room temperature with glutathione sepharose 4B beads conjugated with six times molar excess of ( a ) GST (lane 1), GST-tagged importin- β (lane 2), - α 5 (lane 3), - α 1 (lane 4) and - α 3 (lane 7) or with GST-tagged importin- α 5, α 1, α 3 in presence of HA-tagged importin- β in (lane 4, 6 and 8 respectively). ( b ) GST (lane 1) or GST-importins- α 3/HA-importin- β complex (lane 2) or in presence of peptide SV40 NLS, TG2 NES and TG2 NLS (lane 3, 4 and 5, respectively) ( c ) GST (lane 1) or GST-tagged exportin-1 (Exp-1) in the presence or absence of 0.1% EtOH, 1 mM ACR and 10 μ g/ml LMB as indicated. After spin-down, proteins were eluted with SDS-PAGE sample buffer and TG2 level in each pull down obtained under each condition were determined by western blotting using an antibody against TG2. A representative image, showing intensity value of each blot relative to lane 2 is presented as mean for three independent experiments ( a ) or as mean for two independent experiments ( b and c ). Specificity in the immunoprecipitation experiment of the TG2-exportin-1 complex was ensured in the control experiment ( Supplementary Figure 7 ). ( d ) JHH-7 cells were seeded at 1 × 10 3 cells per well in 96-well plate and incubated at 37 °C overnight. PLA was performed according to the manufacturer's instruction. In control (row 1), the cells were treated with media for 3 h and cells were fixed, permeabilized and no antibodies were used against TG2 and importins for PLA assay. While in others, cells were treated with 0.1% EtOH for 3 h, fixed, permeabilized and treated with combinations of mouse anti-TG2 (CUB7402) and rabbit anti-KPNA4 (left panel, row 2), mouse anti-TG2 (CUB7402) and rabbit anti-SRP-1 (central panel, row 2) or mouse anti-TG2 (CUB7402) and rabbit anti-importin- β (right panel, row 2). After amplification and staining with H33258, the cells were observed under a confocal microscope. Red fluorescence dots derived from amplification of detected protein interaction was monitored with blue fluorescence from H33258. A scale bar=20 μ m. A representative image from three independent experiments with similar results is presented

Techniques Used: Recombinant, Incubation, SDS Page, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Amplification, Staining, Microscopy, Fluorescence, Derivative Assay

Effect of ACR in trimeric complex formation. ( a ) Recombinant human TG2 (1.5 pmol) was incubated for 1 h at room temperature with glutathione sepharose 4B beads conjugated with six times molar excess of GST-importins- α 3/HA-tagged importin- β complex in the presence or absence of ATP, EtOH or ACR as indicated. After spin-down, proteins were eluted with SDS-PAGE sample buffer and TG2 levels in each co-precipitate obtained under each condition were determined by western blotting using an antibody against TG2. A representative result from two independent experiments with similar results is presented. ( b ) JHH-7 cells were seeded at 1 × 10 6 cells per 10-cm dish overnight. The cells were then treated with 0.1% EtOH (column 1 and 3) or 10 μ M ACR (column 2 and 4) for next 5 h. The cells were lysed using Tris buffer (pH 7.4) with 1% Triton X-100, 0.1 mg/ml PMSF and the protease inhibitor cocktail. Importin- β (row 1) and importin- α 3 (row 2) were co-immunoprecipitated using TG2 antibody (CUB7402) from samples containing equal amount of total protein determined by bicinchoninic acid (BCA) protein assay method. After precipitation, proteins were eluted with SDS-PAGE sample buffer and levels of importin- β and - α 3 in each co-precipitation obtained under each condition were determined by western blotting using an antibody indicated. ( c ) JHH-7 cells were seeded at 1 × 10 3 cells per well in 96-well plate and incubated at 37 °C overnight. The cells were then treated with 0.1% EtOH (columns 1–3) or 10 μ M ACR (columns 4–6) for the next 3, 5 and 7 h (rows 1–3). The cells were then fixed and PLA was performed as per manufacturer's instruction. The cells were treated with mouse anti-TG2 (CUB 7402) and rabbit anti-importin- β . After amplification and staining with H33258, the cells were observed under a confocal microscope. Red fluorescence dots derived from amplification of detected protein interaction were monitored with blue fluorescence from H33258. A scale bar, 20 μ m. A representative image from two independent experiments is presented. White arrow heads signify the nuclear TG2-importin β complex.
Figure Legend Snippet: Effect of ACR in trimeric complex formation. ( a ) Recombinant human TG2 (1.5 pmol) was incubated for 1 h at room temperature with glutathione sepharose 4B beads conjugated with six times molar excess of GST-importins- α 3/HA-tagged importin- β complex in the presence or absence of ATP, EtOH or ACR as indicated. After spin-down, proteins were eluted with SDS-PAGE sample buffer and TG2 levels in each co-precipitate obtained under each condition were determined by western blotting using an antibody against TG2. A representative result from two independent experiments with similar results is presented. ( b ) JHH-7 cells were seeded at 1 × 10 6 cells per 10-cm dish overnight. The cells were then treated with 0.1% EtOH (column 1 and 3) or 10 μ M ACR (column 2 and 4) for next 5 h. The cells were lysed using Tris buffer (pH 7.4) with 1% Triton X-100, 0.1 mg/ml PMSF and the protease inhibitor cocktail. Importin- β (row 1) and importin- α 3 (row 2) were co-immunoprecipitated using TG2 antibody (CUB7402) from samples containing equal amount of total protein determined by bicinchoninic acid (BCA) protein assay method. After precipitation, proteins were eluted with SDS-PAGE sample buffer and levels of importin- β and - α 3 in each co-precipitation obtained under each condition were determined by western blotting using an antibody indicated. ( c ) JHH-7 cells were seeded at 1 × 10 3 cells per well in 96-well plate and incubated at 37 °C overnight. The cells were then treated with 0.1% EtOH (columns 1–3) or 10 μ M ACR (columns 4–6) for the next 3, 5 and 7 h (rows 1–3). The cells were then fixed and PLA was performed as per manufacturer's instruction. The cells were treated with mouse anti-TG2 (CUB 7402) and rabbit anti-importin- β . After amplification and staining with H33258, the cells were observed under a confocal microscope. Red fluorescence dots derived from amplification of detected protein interaction were monitored with blue fluorescence from H33258. A scale bar, 20 μ m. A representative image from two independent experiments is presented. White arrow heads signify the nuclear TG2-importin β complex.

Techniques Used: Recombinant, Incubation, SDS Page, Western Blot, Protease Inhibitor, Immunoprecipitation, BIA-KA, Proximity Ligation Assay, Amplification, Staining, Microscopy, Fluorescence, Derivative Assay

10) Product Images from "Identification of Novel MAGE-G1-Interacting Partners in Retinoic Acid-Induced P19 Neuronal Differentiation Using SILAC-Based Proteomics"

Article Title: Identification of Novel MAGE-G1-Interacting Partners in Retinoic Acid-Induced P19 Neuronal Differentiation Using SILAC-Based Proteomics

Journal: Scientific Reports

doi: 10.1038/srep44699

Validation of the interaction between MAGE-G1 and VIME by GST pull-down and co-immunoprecipitation experiments. ( a ) GST or GST-VIME proteins were expressed in Escherichia coli BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-VIME were detected with indicated antibody. Full-length blots are included in a Supplementary Information . ( b ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Vime or pCMV-3 × Flag empty vector plus GFP- Vime expression plasmids. 25 μg of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-VIME (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. ( c ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Vime or pEGFP empty vector plus Flag- Mage - g1 expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. ( d ) Whole cell lysates from RA-treated P19 cells were immunoprecipitated (IP) with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation and 25 μg whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-VIME antibody.
Figure Legend Snippet: Validation of the interaction between MAGE-G1 and VIME by GST pull-down and co-immunoprecipitation experiments. ( a ) GST or GST-VIME proteins were expressed in Escherichia coli BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-VIME were detected with indicated antibody. Full-length blots are included in a Supplementary Information . ( b ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Vime or pCMV-3 × Flag empty vector plus GFP- Vime expression plasmids. 25 μg of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-VIME (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. ( c ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Vime or pEGFP empty vector plus Flag- Mage - g1 expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. ( d ) Whole cell lysates from RA-treated P19 cells were immunoprecipitated (IP) with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation and 25 μg whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-VIME antibody.

Techniques Used: Immunoprecipitation, Purification, Incubation, Transfection, Plasmid Preparation, Expressing, SDS Page, Negative Control

Validation of the interaction between MAGE-G1 and FSCN1 by GST pull-down and co-immunoprecipitation experiments. ( a ) GST or GST-FSCN1 proteins were expressed in Escherichia coli BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-FSCN1 were detected with indicated antibody. Full-length blots are included in a Supplementary Information . ( b ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Fscn1 or pCMV-3 × Flag empty vector plus pEGFP- Fscn1 expression plasmids. 25 μg of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-FSCN1 (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. ( c ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Fscn1 or pEGFP empty vector plus Flag- Mage - g1 expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. ( d ) Whole cell lysates from RA-treated P19 cells were immunoprecipitated with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation (IP) and 25 μg whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-FSCN1 antibody.
Figure Legend Snippet: Validation of the interaction between MAGE-G1 and FSCN1 by GST pull-down and co-immunoprecipitation experiments. ( a ) GST or GST-FSCN1 proteins were expressed in Escherichia coli BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-FSCN1 were detected with indicated antibody. Full-length blots are included in a Supplementary Information . ( b ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Fscn1 or pCMV-3 × Flag empty vector plus pEGFP- Fscn1 expression plasmids. 25 μg of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-FSCN1 (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. ( c ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Fscn1 or pEGFP empty vector plus Flag- Mage - g1 expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. ( d ) Whole cell lysates from RA-treated P19 cells were immunoprecipitated with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation (IP) and 25 μg whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-FSCN1 antibody.

Techniques Used: Immunoprecipitation, Purification, Incubation, Transfection, Plasmid Preparation, Expressing, SDS Page, Negative Control

11) Product Images from "Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA *"

Article Title: Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.154831

TDP-43 interacts with PABP2 and FUS/TLS. A , identification of TDP-43 interacting proteins by mass spectrometry. HeLa cells transfected with HA-TDP-43 were immunoprecipitated with α-HA-conjugated agarose, and the immunoprecipitated proteins were separated by SDS-PAGE. The gel was stained with Colloidal Blue. Candidate TDP-43-associated proteins were analyzed by mass spectrometry. B , co-IP of HA-TDP-43 with endogenous FUS/TLS and PABP2. HA-TDP-43 was immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA, α-FUS/TLS, and α-PABP2 antibodies. C , co-IP of HA-FUS/TLS and endogenous TDP-43. HeLa cells transfected with HA-FUS/TLS were immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA and α-TDP-43 antibodies. D , interaction of endogenous TDP-43 and FUS/TLS. Exponentially growing HeLa cells were immunoprecipitated with α-FUS/TLS antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with α-FUS/TLS and α-TDP-43 antibodies. E , HA-TDP-43 interacts with GST-FUS/TLS in vitro . GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with HEK 293T cell extract containing HA-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HA antibodies. F , interaction of purified proteins. Purified GST and GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with purified HIS-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HIS antibodies.
Figure Legend Snippet: TDP-43 interacts with PABP2 and FUS/TLS. A , identification of TDP-43 interacting proteins by mass spectrometry. HeLa cells transfected with HA-TDP-43 were immunoprecipitated with α-HA-conjugated agarose, and the immunoprecipitated proteins were separated by SDS-PAGE. The gel was stained with Colloidal Blue. Candidate TDP-43-associated proteins were analyzed by mass spectrometry. B , co-IP of HA-TDP-43 with endogenous FUS/TLS and PABP2. HA-TDP-43 was immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA, α-FUS/TLS, and α-PABP2 antibodies. C , co-IP of HA-FUS/TLS and endogenous TDP-43. HeLa cells transfected with HA-FUS/TLS were immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA and α-TDP-43 antibodies. D , interaction of endogenous TDP-43 and FUS/TLS. Exponentially growing HeLa cells were immunoprecipitated with α-FUS/TLS antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with α-FUS/TLS and α-TDP-43 antibodies. E , HA-TDP-43 interacts with GST-FUS/TLS in vitro . GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with HEK 293T cell extract containing HA-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HA antibodies. F , interaction of purified proteins. Purified GST and GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with purified HIS-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HIS antibodies.

Techniques Used: Mass Spectrometry, Transfection, Immunoprecipitation, SDS Page, Staining, Co-Immunoprecipitation Assay, In Vitro, Incubation, Purification

The Gly-rich and RRM2 domains of TDP-43 contribute to FUS/TLS binding. A , stick diagrams of TDP-43 deletion mutants used in the GST-FUS/TLS pulldown assays. B , GST-FUS/TLS pulldown assay using C-terminal deletion mutants of TDP-43. HA-tagged wild-type TDP-43 or deletion mutants of TDP-43 were expressed in HEK 293T cells, and the cell lysates were incubated with GST or GST-FUS/TLS proteins conjugated to glutathione-Sepharose 4B beads. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with α-GST and α-HA antibodies. C , interaction of FUS/TLS with N-terminal TDP-43 truncation mutants. The indicated TDP-43 N-terminal truncation mutants were expressed in HEK 293T cell and tested for interaction with GST-FUS/TLS in GST pulldown assays. These findings demonstrate that a region spanning amino acids 170–414 of TDP-43 is sufficient for binding to GST-FUS/TLS in vitro. vec , vector; wt , wild type.
Figure Legend Snippet: The Gly-rich and RRM2 domains of TDP-43 contribute to FUS/TLS binding. A , stick diagrams of TDP-43 deletion mutants used in the GST-FUS/TLS pulldown assays. B , GST-FUS/TLS pulldown assay using C-terminal deletion mutants of TDP-43. HA-tagged wild-type TDP-43 or deletion mutants of TDP-43 were expressed in HEK 293T cells, and the cell lysates were incubated with GST or GST-FUS/TLS proteins conjugated to glutathione-Sepharose 4B beads. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with α-GST and α-HA antibodies. C , interaction of FUS/TLS with N-terminal TDP-43 truncation mutants. The indicated TDP-43 N-terminal truncation mutants were expressed in HEK 293T cell and tested for interaction with GST-FUS/TLS in GST pulldown assays. These findings demonstrate that a region spanning amino acids 170–414 of TDP-43 is sufficient for binding to GST-FUS/TLS in vitro. vec , vector; wt , wild type.

Techniques Used: Binding Assay, Incubation, SDS Page, In Vitro, Plasmid Preparation

Wild-type and ALS-associated TDP-43 mutants interact with FUS/TLS comparably. A , co-immunoprecipitation assay. HeLa cells were transfected with HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants. Wild-type and mutant HA-TDP-43 proteins were immunoprecipitated with α-HA. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. B , reciprocal co-immunoprecipitation assay. HeLa cells were transfected with plasmids encoding HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants, and cell extracts were immunoprecipitated with α-FUS/TLS antibodies. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. C , GST-FUS/TLS pulldown of ALS-associated TDP-43 mutants. GST and GST-FUS/TLS were induced in BL 21 cells and purified using glutathione-Sepharose 4B beads. HA-tagged wild-type TDP-43 or ALS-associated mutants of TDP-43 were expressed in HEK 293T cells, and corresponding cell extracts were incubated with GST or GST-FUS/TLS. The affinity-purified proteins were separated by 10% SDS-PAGE and analyzed by Western blotting with α-GST and α-HA antibodies. vec , vector.
Figure Legend Snippet: Wild-type and ALS-associated TDP-43 mutants interact with FUS/TLS comparably. A , co-immunoprecipitation assay. HeLa cells were transfected with HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants. Wild-type and mutant HA-TDP-43 proteins were immunoprecipitated with α-HA. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. B , reciprocal co-immunoprecipitation assay. HeLa cells were transfected with plasmids encoding HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants, and cell extracts were immunoprecipitated with α-FUS/TLS antibodies. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. C , GST-FUS/TLS pulldown of ALS-associated TDP-43 mutants. GST and GST-FUS/TLS were induced in BL 21 cells and purified using glutathione-Sepharose 4B beads. HA-tagged wild-type TDP-43 or ALS-associated mutants of TDP-43 were expressed in HEK 293T cells, and corresponding cell extracts were incubated with GST or GST-FUS/TLS. The affinity-purified proteins were separated by 10% SDS-PAGE and analyzed by Western blotting with α-GST and α-HA antibodies. vec , vector.

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Immunoprecipitation, Purification, Incubation, Affinity Purification, SDS Page, Western Blot, Plasmid Preparation

12) Product Images from "Physical and functional interaction between DDB and XPA in nucleotide excision repair"

Article Title: Physical and functional interaction between DDB and XPA in nucleotide excision repair

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn964

DDB directly binds to XPA through DDB2 subunit. Purified DDB heterodimer ( A ) or each DDB subunit ( B ) was incubated with GST alone (lane 2) or GST-XPA (lane 3) coupled to glutathione–sepharose 4B beads. The bound proteins were separated on a SDS–polyacrylamide gel and analyzed by western blotting with anti-Flag antibody for detecting DDB1 and DDB2. ( C ) Purified (His) 6 -XPA protein was incubated with MBP alone (lane 2) or MBP-DDB2 (lane 3) coupled to amylose beads. The bound proteins were analyzed by western blotting with anti-His antibody.
Figure Legend Snippet: DDB directly binds to XPA through DDB2 subunit. Purified DDB heterodimer ( A ) or each DDB subunit ( B ) was incubated with GST alone (lane 2) or GST-XPA (lane 3) coupled to glutathione–sepharose 4B beads. The bound proteins were separated on a SDS–polyacrylamide gel and analyzed by western blotting with anti-Flag antibody for detecting DDB1 and DDB2. ( C ) Purified (His) 6 -XPA protein was incubated with MBP alone (lane 2) or MBP-DDB2 (lane 3) coupled to amylose beads. The bound proteins were analyzed by western blotting with anti-His antibody.

Techniques Used: Purification, Incubation, Western Blot

R207G mutation reduces XPA binding to DDB. ( A ) Purified DDB heterodimer or in vitro translated ERCC1 was incubated with GST alone (lane 2), GST-XPA (lane 3) or GST-XPA(R207G) (lane 4) coupled to glutathione–sepharose 4B beads. The bound proteins were analyzed by western blotting with either anti-Flag followed by anti-mouse IgG conjugated with alkaline phosphatase (for DDB1 and DDB2) or streptavidin conjugated with alkaline phosphatase (for ERCC1). ( B) and ( C ) myc-tagged XPA protein, wild-type or R207G mutant, was expressed in Tet-on U2OS/3xF-DDB2 cells in the presence of doxycycline and cell lysates were prepared after 40-h incubation. One or 0.3 mg of the lysates were incubated for 1.5 h with anti-FLAG M2 agarose (B) or anti-myc antibody followed by protein A/G plus agarose (C), respectively. Proteins retained on the beads were analyzed by western blotting using anti-Flag and anti-myc antibodies.
Figure Legend Snippet: R207G mutation reduces XPA binding to DDB. ( A ) Purified DDB heterodimer or in vitro translated ERCC1 was incubated with GST alone (lane 2), GST-XPA (lane 3) or GST-XPA(R207G) (lane 4) coupled to glutathione–sepharose 4B beads. The bound proteins were analyzed by western blotting with either anti-Flag followed by anti-mouse IgG conjugated with alkaline phosphatase (for DDB1 and DDB2) or streptavidin conjugated with alkaline phosphatase (for ERCC1). ( B) and ( C ) myc-tagged XPA protein, wild-type or R207G mutant, was expressed in Tet-on U2OS/3xF-DDB2 cells in the presence of doxycycline and cell lysates were prepared after 40-h incubation. One or 0.3 mg of the lysates were incubated for 1.5 h with anti-FLAG M2 agarose (B) or anti-myc antibody followed by protein A/G plus agarose (C), respectively. Proteins retained on the beads were analyzed by western blotting using anti-Flag and anti-myc antibodies.

Techniques Used: Mutagenesis, Binding Assay, Purification, In Vitro, Incubation, Western Blot

Domain mapping of XPA responsible for the binding to DDB2. ( A ) Schematic diagram of various XPA deletion mutants and summary of pull-down experiments with these mutants. ( B ) The lysates from insect cells overproducing DDB2 were incubated with GST alone (lane 1) or various GST-XPA derivatives (lanes 2–7) coupled to glutathione–sepharose 4B beads. The bound proteins were analyzed by western blotting with anti-Flag antibody.
Figure Legend Snippet: Domain mapping of XPA responsible for the binding to DDB2. ( A ) Schematic diagram of various XPA deletion mutants and summary of pull-down experiments with these mutants. ( B ) The lysates from insect cells overproducing DDB2 were incubated with GST alone (lane 1) or various GST-XPA derivatives (lanes 2–7) coupled to glutathione–sepharose 4B beads. The bound proteins were analyzed by western blotting with anti-Flag antibody.

Techniques Used: Binding Assay, Incubation, Western Blot

13) Product Images from "The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment"

Article Title: The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg251

Fig. 3. VPg interactions with eIF4GI and other initiation factors. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. Western blots were probed with ( A ) anti-eIF4GI, or ( B ) anti-eIF2α, anti-eIF4E, anti-S6 or anti-eIF4B.
Figure Legend Snippet: Fig. 3. VPg interactions with eIF4GI and other initiation factors. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. Western blots were probed with ( A ) anti-eIF4GI, or ( B ) anti-eIF2α, anti-eIF4E, anti-S6 or anti-eIF4B.

Techniques Used: Mutagenesis, Construct, Incubation, Western Blot

Fig. 2. VPg binds eIF3 present in cell lysates. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. ( A ) CaCo-2 cell lysates. ( B ) S7-treated CaCo-2 lysates. ( C ) GST–SMV-VPg immobilized to glutathione–Sepharose beads. ( D ) VPg deletion mutants. Western blots were probed with anti-eIF3 polyclonal antibody. Asterisks indicate a protein likely to be eIF4GI.
Figure Legend Snippet: Fig. 2. VPg binds eIF3 present in cell lysates. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. ( A ) CaCo-2 cell lysates. ( B ) S7-treated CaCo-2 lysates. ( C ) GST–SMV-VPg immobilized to glutathione–Sepharose beads. ( D ) VPg deletion mutants. Western blots were probed with anti-eIF3 polyclonal antibody. Asterisks indicate a protein likely to be eIF4GI.

Techniques Used: Mutagenesis, Construct, Incubation, Western Blot

Fig. 1. VPg binds initiation factor eIF3. Pull-down assays were conducted with GST or GST–VPg immobilized on glutathione–Sepharose 4B beads. ( A ) Pull-down with purified eIF3. The input eIF3 lane represents 10% of the amount of protein used in the pull-down reaction, and the western blot was probed with anti-eIF3 polyclonal antibody. ( B ) Pull-down with 35 S-labeled in vitro translated subunit eIF3d. The input eIF3d lane represents 25% of the amount used in the GST and GST–VPg pull-downs. Pull-down eluates were separated by 10% SDS–PAGE and exposed to film for autoradiography.
Figure Legend Snippet: Fig. 1. VPg binds initiation factor eIF3. Pull-down assays were conducted with GST or GST–VPg immobilized on glutathione–Sepharose 4B beads. ( A ) Pull-down with purified eIF3. The input eIF3 lane represents 10% of the amount of protein used in the pull-down reaction, and the western blot was probed with anti-eIF3 polyclonal antibody. ( B ) Pull-down with 35 S-labeled in vitro translated subunit eIF3d. The input eIF3d lane represents 25% of the amount used in the GST and GST–VPg pull-downs. Pull-down eluates were separated by 10% SDS–PAGE and exposed to film for autoradiography.

Techniques Used: Purification, Western Blot, Labeling, In Vitro, SDS Page, Autoradiography

14) Product Images from "Deacetylation-mediated interaction of SIRT1-HMGB1 improves survival in a mouse model of endotoxemia"

Article Title: Deacetylation-mediated interaction of SIRT1-HMGB1 improves survival in a mouse model of endotoxemia

Journal: Scientific Reports

doi: 10.1038/srep15971

LPS promotes HMGB1 release via its dissociation from SIRT1. ( A–C ) HEK293T cells co-transfected with Flag-HMGB1 and/or Myc-SIRT1 for 48 h were incubated with LPS (100 ng/ml), Poly (I:C) (50 μg/ml), IFN-γ (40 ng/ml), or TNF-α (20 ng/ml) for 3 h, and then whole-cell lysates were immunoprecipitated with an anti-Flag ( A,C ) or anti-Myc ( B ) antibody and analyzed by Western blotting. ( D,E ) HEK293T cells expressing Flag-HMGB1 were incubated with or without the indicated stimuli for 3 h. Whole-cell lysates were incubated with recombinant GST or GST-SIRT1 fusion protein immobilized to glutathione-Sepharose 4B beads for 20 h, and then pulled down or immunoprecipitated. GST and GST-fused proteins were stained with Ponceau S. ( F ) RAW 264.7 cells co-transfected with Flag-HMGB1 and Myc-SIRT1 for 48 h were incubated with or without LPS (100 ng/ml) for 6 h (for interaction) or 24 h (for HMGB1 release). Whole-cell lysates were immunoprecipitated with an anti-HMGB1 antibody to determine the interaction with SIRT1. To detect released HMGB1, equal volumes of conditioned media were analyzed by Western blotting.
Figure Legend Snippet: LPS promotes HMGB1 release via its dissociation from SIRT1. ( A–C ) HEK293T cells co-transfected with Flag-HMGB1 and/or Myc-SIRT1 for 48 h were incubated with LPS (100 ng/ml), Poly (I:C) (50 μg/ml), IFN-γ (40 ng/ml), or TNF-α (20 ng/ml) for 3 h, and then whole-cell lysates were immunoprecipitated with an anti-Flag ( A,C ) or anti-Myc ( B ) antibody and analyzed by Western blotting. ( D,E ) HEK293T cells expressing Flag-HMGB1 were incubated with or without the indicated stimuli for 3 h. Whole-cell lysates were incubated with recombinant GST or GST-SIRT1 fusion protein immobilized to glutathione-Sepharose 4B beads for 20 h, and then pulled down or immunoprecipitated. GST and GST-fused proteins were stained with Ponceau S. ( F ) RAW 264.7 cells co-transfected with Flag-HMGB1 and Myc-SIRT1 for 48 h were incubated with or without LPS (100 ng/ml) for 6 h (for interaction) or 24 h (for HMGB1 release). Whole-cell lysates were immunoprecipitated with an anti-HMGB1 antibody to determine the interaction with SIRT1. To detect released HMGB1, equal volumes of conditioned media were analyzed by Western blotting.

Techniques Used: Transfection, Incubation, Immunoprecipitation, Western Blot, Expressing, Recombinant, Staining

HMGB1 directly interacts with SIRT1. ( A , B ) HEK293T cells were co-transfected with the indicated plasmids for 48 h, and then whole-cell lysates were prepared and immunoprecipitated with IgG, anti-Flag, or anti-Myc antibody. The immunoprecipitates and total lysates (input) were subjected to immunoblot analysis with anti-Flag, anti-Myc, and anti-β-actin antibodies to detect HMGB1, SIRT1, and β-actin, respectively. Two percent of whole-cell lysates were used as the input. ( C ) HEK293T cells were transfected with Flag-tagged HMGB1 for 48 h, and whole-cell lysates were incubated with recombinant GST or GST-SIRT1 fusion protein immobilized to glutathione-Sepharose 4B beads for 20 h. Bead-bound proteins were analyzed by Western blotting. GST and GST-fused proteins were stained with Ponceau S. ( D ) The fluorescence of each fusion protein was visualized in HEK293T cells by confocal microscopy. The co-localization of HMGB1 and SIRT1 is indicated by the presence of yellow in the merge image. The bar indicates 10 μm. ( E ) Constructs of Flag-tagged HMGB1 are schematically illustrated. ( F , G ) HEK293T cells were co-transfected with Myc-SIRT1 and plasmids harboring Flag-HMGB1 FL or deletion mutants for 48 h. Whole-cell lysates were immunoprecipitated with an anti-Flag antibody. ( H ) HEK293T cells were transfected with Flag-tagged HMGB1 deletion mutants for 48 h. Whole-cell lysates were then prepared and incubated with recombinant GST or GST-SIRT1 fusion protein immobilized to glutathione-Sepharose 4B beads for 20 h. Bead-bound proteins were analyzed by Western blotting. GST and GST-fused proteins were stained with Ponceau S.
Figure Legend Snippet: HMGB1 directly interacts with SIRT1. ( A , B ) HEK293T cells were co-transfected with the indicated plasmids for 48 h, and then whole-cell lysates were prepared and immunoprecipitated with IgG, anti-Flag, or anti-Myc antibody. The immunoprecipitates and total lysates (input) were subjected to immunoblot analysis with anti-Flag, anti-Myc, and anti-β-actin antibodies to detect HMGB1, SIRT1, and β-actin, respectively. Two percent of whole-cell lysates were used as the input. ( C ) HEK293T cells were transfected with Flag-tagged HMGB1 for 48 h, and whole-cell lysates were incubated with recombinant GST or GST-SIRT1 fusion protein immobilized to glutathione-Sepharose 4B beads for 20 h. Bead-bound proteins were analyzed by Western blotting. GST and GST-fused proteins were stained with Ponceau S. ( D ) The fluorescence of each fusion protein was visualized in HEK293T cells by confocal microscopy. The co-localization of HMGB1 and SIRT1 is indicated by the presence of yellow in the merge image. The bar indicates 10 μm. ( E ) Constructs of Flag-tagged HMGB1 are schematically illustrated. ( F , G ) HEK293T cells were co-transfected with Myc-SIRT1 and plasmids harboring Flag-HMGB1 FL or deletion mutants for 48 h. Whole-cell lysates were immunoprecipitated with an anti-Flag antibody. ( H ) HEK293T cells were transfected with Flag-tagged HMGB1 deletion mutants for 48 h. Whole-cell lysates were then prepared and incubated with recombinant GST or GST-SIRT1 fusion protein immobilized to glutathione-Sepharose 4B beads for 20 h. Bead-bound proteins were analyzed by Western blotting. GST and GST-fused proteins were stained with Ponceau S.

Techniques Used: Transfection, Immunoprecipitation, Incubation, Recombinant, Western Blot, Staining, Fluorescence, Confocal Microscopy, Construct

15) Product Images from "Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication"

Article Title: Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication

Journal: Scientific Reports

doi: 10.1038/srep35041

NS1 interacts with Rac1 in vivo . ( A ) GST-pull down assay showing the interaction between NS1 and Rac1. Whole 293T cell lysates transfected with Myc-tagged Rac1 were incubated with an equal amount of GST or GST-NS1 bound to glutathione-Sepharose 4B beads, followed by IB using the anti-Myc antibody. CBB, Coomassie brilliant blue staining. ( B ) Co-immunoprecipitation of Flag-NS1 and Myc-Rac1 in 293T cells. The 293T cells were co-transfected with Flag-tagged NS1 and pcDNA4.0 or pcDNA4.0-myc-Rac1; Myc-tagged Rac1 and pcDNA3.0 or pcDNA3.0-flag-NS1. Rac1 antibodies (rabbit) were applied to the cell lysates for IP, followed by IB with Flag (mouse); alternatively, the IP was performed with a Flag antibody (mouse) followed by IB with Rac1 (rabbit). ( C ) Co-localization of GFP-NS1 and Myc-Rac1 in A549 cells. A549 cells were co-transfected with pEGFP-NS1 and pcDNA4.0-myc-Rac1 for 28 hours and then fixed, permeabilized, and stained for Myc-Rac1 (red). Yellow indicates overlap. ( D ) Co-immunoprecipitation of virus NS1 and endogenous Rac1 in A549 cells. A549 cells were infected with the A/WSN/33 H1N1 virus and the mutated NS1 − H1N1 virus (MOI = 0.5) for 24 hours, and the cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. ( E ) Co-localization of viral NS1 and endogenous Rac1 in A549 cells. A549 cells were infected with the A/WSN/33 H1N1 virus (MOI = 0.1) for 24 hours and then fixed, permeabilized, and stained with DAPI (blue), anti-NS1 antibody (red), and anti-Rac1 antibody (green). Yellow indicates overlap with red and green. Orange indicates overlap with blue, red and green.
Figure Legend Snippet: NS1 interacts with Rac1 in vivo . ( A ) GST-pull down assay showing the interaction between NS1 and Rac1. Whole 293T cell lysates transfected with Myc-tagged Rac1 were incubated with an equal amount of GST or GST-NS1 bound to glutathione-Sepharose 4B beads, followed by IB using the anti-Myc antibody. CBB, Coomassie brilliant blue staining. ( B ) Co-immunoprecipitation of Flag-NS1 and Myc-Rac1 in 293T cells. The 293T cells were co-transfected with Flag-tagged NS1 and pcDNA4.0 or pcDNA4.0-myc-Rac1; Myc-tagged Rac1 and pcDNA3.0 or pcDNA3.0-flag-NS1. Rac1 antibodies (rabbit) were applied to the cell lysates for IP, followed by IB with Flag (mouse); alternatively, the IP was performed with a Flag antibody (mouse) followed by IB with Rac1 (rabbit). ( C ) Co-localization of GFP-NS1 and Myc-Rac1 in A549 cells. A549 cells were co-transfected with pEGFP-NS1 and pcDNA4.0-myc-Rac1 for 28 hours and then fixed, permeabilized, and stained for Myc-Rac1 (red). Yellow indicates overlap. ( D ) Co-immunoprecipitation of virus NS1 and endogenous Rac1 in A549 cells. A549 cells were infected with the A/WSN/33 H1N1 virus and the mutated NS1 − H1N1 virus (MOI = 0.5) for 24 hours, and the cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. ( E ) Co-localization of viral NS1 and endogenous Rac1 in A549 cells. A549 cells were infected with the A/WSN/33 H1N1 virus (MOI = 0.1) for 24 hours and then fixed, permeabilized, and stained with DAPI (blue), anti-NS1 antibody (red), and anti-Rac1 antibody (green). Yellow indicates overlap with red and green. Orange indicates overlap with blue, red and green.

Techniques Used: In Vivo, Pull Down Assay, Transfection, Incubation, Staining, Immunoprecipitation, Infection

NS1 down-regulates Rac1 activity. ( A ) There was no significant difference in the Rac1 protein expression level between the two different cell lines. The two cell lines were seeded into six-well plates. After 24 hours, the Rac1 protein expression levels were assessed in the whole cell lysates using the indicated antibodies as appropriate. ( B ) GTP-bound Rac1 levels were lower in the NS1 over-expressing cell lines than in the negative control cell lines. Lysates from the two stable cell lines were incubated with equal amounts of GST-PAK bound glutathione Sepharose 4B beads. After washing, the bound proteins were analyzed by western blotting using an anti-Rac1 antibody. The protein band intensity was measured using the ImageJ software (NIH). Data represent the mean fold change ± S.D. of three independent experiments (*p
Figure Legend Snippet: NS1 down-regulates Rac1 activity. ( A ) There was no significant difference in the Rac1 protein expression level between the two different cell lines. The two cell lines were seeded into six-well plates. After 24 hours, the Rac1 protein expression levels were assessed in the whole cell lysates using the indicated antibodies as appropriate. ( B ) GTP-bound Rac1 levels were lower in the NS1 over-expressing cell lines than in the negative control cell lines. Lysates from the two stable cell lines were incubated with equal amounts of GST-PAK bound glutathione Sepharose 4B beads. After washing, the bound proteins were analyzed by western blotting using an anti-Rac1 antibody. The protein band intensity was measured using the ImageJ software (NIH). Data represent the mean fold change ± S.D. of three independent experiments (*p

Techniques Used: Activity Assay, Expressing, Negative Control, Stable Transfection, Incubation, Western Blot, Software

16) Product Images from "Direct Interaction with Rab11a Targets the Epithelial Ca2+ Channels TRPV5 and TRPV6 to the Plasma Membrane"

Article Title: Direct Interaction with Rab11a Targets the Epithelial Ca2+ Channels TRPV5 and TRPV6 to the Plasma Membrane

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.26.1.303-312.2006

Rab11a-binding site in TRPV5. (A) GST fusion proteins containing different portions of the carboxyl terminus of mouse TRPV5 were constructed according to the schematic drawing. (B) These proteins were immobilized on glutathione-Sepharose 4B beads and then incubated with in vitro-translated Rab11a S25N. Interaction of Rab11a with the GST fusion proteins was determined by autoradiography. The region between amino acids 595 and 601 was essential for binding. Mutation of this region (595-5G-601) diminished the interaction with Rab11a (right). (C) GST or GST fused to the carboxyl terminus of TRPV5 and immobilized on glutathione-Sepharose 4B beads was incubated with in vitro-translated Rab22b; precipitated proteins were analyzed by autoradiography. Rab22b did not associate with TRPV5, demonstrating the specificity of the Rab11a binding. (D) TRPV5 and TRPV6 sequences, varying from humans to zebra fish, were aligned by the Clustal method, demonstrating complete conservation of the Rab11a-binding site during evolution. (E) cRNA encoding full-length TRPV5 or TRPV6 was injected in Xenopus laevis oocytes, and the localization of the channel was investigated by immunocytochemistry. Mutation of five amino acids in the Rab11a-binding region resulted in significantly disturbed TRPV5/6 trafficking, resulting in a small number of channels at the cell surface. (F) Accordingly, TRPV5-mediated 45 Ca 2+ uptake was significantly decreased in Xenopus laevis oocytes injected with TRPV5 595-5G-601, compared to oocytes injected with wild-type TRPV5. Similar functional results were obtained between the wild type and 600-5G-607 mutant TRPV6. Bar, 25 μm.
Figure Legend Snippet: Rab11a-binding site in TRPV5. (A) GST fusion proteins containing different portions of the carboxyl terminus of mouse TRPV5 were constructed according to the schematic drawing. (B) These proteins were immobilized on glutathione-Sepharose 4B beads and then incubated with in vitro-translated Rab11a S25N. Interaction of Rab11a with the GST fusion proteins was determined by autoradiography. The region between amino acids 595 and 601 was essential for binding. Mutation of this region (595-5G-601) diminished the interaction with Rab11a (right). (C) GST or GST fused to the carboxyl terminus of TRPV5 and immobilized on glutathione-Sepharose 4B beads was incubated with in vitro-translated Rab22b; precipitated proteins were analyzed by autoradiography. Rab22b did not associate with TRPV5, demonstrating the specificity of the Rab11a binding. (D) TRPV5 and TRPV6 sequences, varying from humans to zebra fish, were aligned by the Clustal method, demonstrating complete conservation of the Rab11a-binding site during evolution. (E) cRNA encoding full-length TRPV5 or TRPV6 was injected in Xenopus laevis oocytes, and the localization of the channel was investigated by immunocytochemistry. Mutation of five amino acids in the Rab11a-binding region resulted in significantly disturbed TRPV5/6 trafficking, resulting in a small number of channels at the cell surface. (F) Accordingly, TRPV5-mediated 45 Ca 2+ uptake was significantly decreased in Xenopus laevis oocytes injected with TRPV5 595-5G-601, compared to oocytes injected with wild-type TRPV5. Similar functional results were obtained between the wild type and 600-5G-607 mutant TRPV6. Bar, 25 μm.

Techniques Used: Binding Assay, Construct, Incubation, In Vitro, Autoradiography, Mutagenesis, Fluorescence In Situ Hybridization, Injection, Immunocytochemistry, Functional Assay

Interaction of TRPV5 and TRPV6 with Rab11a. (A) The carboxyl terminus of TRPV6 or γENaC and full-length Rab11a were cotransformed into the Y153 yeast strain and grown on media without tryptophan and leucine. β-Galactosidase activity was determined for TRPV6- and Rab11a-cotransformed yeast, and γENaC- and Rab11a-cotransformed yeast was used as a negative control. Two representative colonies are depicted. (B) [ 35 S]methionine-labeled TRPV5 (left) or TRPV6 (right) was incubated with GST-Rab11a WT, GTP-locked mutant Rab11a (S20V), GDP-locked Rab11a (S25N), or GST alone immobilized on glutathione-Sepharose 4B beads. (C) Coomassie staining of the gels shows equal expression of GST, GST-fused Rab11a WT S20V, or S25N. (D) [ 35 S]methionine-labeled Rab11a S20V or Rab11a S25N was incubated with GST-TRPV5, GST-TRPV6, or GST immobilized on glutathione-Sepharose 4B beads. Both TRPV5 and TRPV6 strongly interacted with Rab11a S25N, whereas no binding to Rab11a S20V was observed. (E) Coomassie staining of the gels demonstrating similar amounts of GST, GST fused to the carboxyl terminus of TRPV5, or GST fused to the carboxyl terminus of TRPV6. (F and G) Xenopus laevis oocytes were injected with Rab11a S25N or coinjected with HA-tagged TRPV5 and Rab11a S25N cRNAs. Oocyte lysates were subjected to immunoprecipitation using monoclonal anti-HA antibodies. The precipitated sample was immunoblotted for the presence of TRPV5 (F) or coprecipitated Rab11a (G).
Figure Legend Snippet: Interaction of TRPV5 and TRPV6 with Rab11a. (A) The carboxyl terminus of TRPV6 or γENaC and full-length Rab11a were cotransformed into the Y153 yeast strain and grown on media without tryptophan and leucine. β-Galactosidase activity was determined for TRPV6- and Rab11a-cotransformed yeast, and γENaC- and Rab11a-cotransformed yeast was used as a negative control. Two representative colonies are depicted. (B) [ 35 S]methionine-labeled TRPV5 (left) or TRPV6 (right) was incubated with GST-Rab11a WT, GTP-locked mutant Rab11a (S20V), GDP-locked Rab11a (S25N), or GST alone immobilized on glutathione-Sepharose 4B beads. (C) Coomassie staining of the gels shows equal expression of GST, GST-fused Rab11a WT S20V, or S25N. (D) [ 35 S]methionine-labeled Rab11a S20V or Rab11a S25N was incubated with GST-TRPV5, GST-TRPV6, or GST immobilized on glutathione-Sepharose 4B beads. Both TRPV5 and TRPV6 strongly interacted with Rab11a S25N, whereas no binding to Rab11a S20V was observed. (E) Coomassie staining of the gels demonstrating similar amounts of GST, GST fused to the carboxyl terminus of TRPV5, or GST fused to the carboxyl terminus of TRPV6. (F and G) Xenopus laevis oocytes were injected with Rab11a S25N or coinjected with HA-tagged TRPV5 and Rab11a S25N cRNAs. Oocyte lysates were subjected to immunoprecipitation using monoclonal anti-HA antibodies. The precipitated sample was immunoblotted for the presence of TRPV5 (F) or coprecipitated Rab11a (G).

Techniques Used: Activity Assay, Negative Control, Labeling, Incubation, Mutagenesis, Staining, Expressing, Binding Assay, Injection, Immunoprecipitation

17) Product Images from "Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain"

Article Title: Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain

Journal: Open Biology

doi: 10.1098/rsob.160089

Details of the molecular recognition in the Aurora-A/vNAR-D01 complex. ( a ) Key interactions are shown in the three panels. Aurora-A is coloured teal and vNAR-D01 is coloured orange. ( b ) Co-precipitation assay between GST-Aurora-A KD DN and WT, and mutant vNAR-D01 constructs. GST-Aurora-A KD DN was immobilized on Glutathione Sepharose 4B beads and then incubated with vNAR-D01 proteins. GST was used as a binding control.
Figure Legend Snippet: Details of the molecular recognition in the Aurora-A/vNAR-D01 complex. ( a ) Key interactions are shown in the three panels. Aurora-A is coloured teal and vNAR-D01 is coloured orange. ( b ) Co-precipitation assay between GST-Aurora-A KD DN and WT, and mutant vNAR-D01 constructs. GST-Aurora-A KD DN was immobilized on Glutathione Sepharose 4B beads and then incubated with vNAR-D01 proteins. GST was used as a binding control.

Techniques Used: Mutagenesis, Construct, Incubation, Binding Assay

vNAR-D01 is an Aurora-A inhibitor that competes with TPX2. ( a ) Surface plasmon resonance binding assays between Aurora-A KD CA-Avi and vNAR-D01. The kinase was immobilized on Biacore Sensor SA chips at 550, 350 and 250 RU and interacted with 0.01–50 µM vNAR-D01. Maximum responses were plotted against vNAR-D01 concentration and fitted to a one-site specific binding equation (solid lines) in P rism 6 (GraphPad) to calculate binding affinities. ( b ) Co-precipitation assay between the Aurora-A KD CA/vNAR-D01 complex or His 6 -Aurora-A KD CA and GST-TPX2 1–43 . The complex and Aurora-A were immobilized on Nickel Sepharose beads using the His 6 -tag on the vNAR domain and kinase, respectively. GST was used as a binding control. ( c ) Co-precipitation assay between GST-Aurora-A KD DN and vNAR-D01 and His 6 -TPX2 1–43 . In total, 2 µM GST-Aurora-A KD DN was immobilized on Glutathione Sepharose 4B beads and incubated with 5 µM vNAR-D01 and 0, 1, 2, 5, 10, 20 and 50 µM His 6 -TPX2 (black triangle). GST was used as a binding control. ( d ) In vitro kinase activity assay of Aurora-A KD in the presence of vNAR-D01. MBP was used as a generic kinase substrate. Reactions were analysed by SDS-PAGE (top left panel) and incorporation of radioisotope resolved by autoradiography (bottom left panel). Incorporation of radioisotope was measured by scintillation counting (right). Error bars represent the standard error for two independent reactions. ** = p
Figure Legend Snippet: vNAR-D01 is an Aurora-A inhibitor that competes with TPX2. ( a ) Surface plasmon resonance binding assays between Aurora-A KD CA-Avi and vNAR-D01. The kinase was immobilized on Biacore Sensor SA chips at 550, 350 and 250 RU and interacted with 0.01–50 µM vNAR-D01. Maximum responses were plotted against vNAR-D01 concentration and fitted to a one-site specific binding equation (solid lines) in P rism 6 (GraphPad) to calculate binding affinities. ( b ) Co-precipitation assay between the Aurora-A KD CA/vNAR-D01 complex or His 6 -Aurora-A KD CA and GST-TPX2 1–43 . The complex and Aurora-A were immobilized on Nickel Sepharose beads using the His 6 -tag on the vNAR domain and kinase, respectively. GST was used as a binding control. ( c ) Co-precipitation assay between GST-Aurora-A KD DN and vNAR-D01 and His 6 -TPX2 1–43 . In total, 2 µM GST-Aurora-A KD DN was immobilized on Glutathione Sepharose 4B beads and incubated with 5 µM vNAR-D01 and 0, 1, 2, 5, 10, 20 and 50 µM His 6 -TPX2 (black triangle). GST was used as a binding control. ( d ) In vitro kinase activity assay of Aurora-A KD in the presence of vNAR-D01. MBP was used as a generic kinase substrate. Reactions were analysed by SDS-PAGE (top left panel) and incorporation of radioisotope resolved by autoradiography (bottom left panel). Incorporation of radioisotope was measured by scintillation counting (right). Error bars represent the standard error for two independent reactions. ** = p

Techniques Used: SPR Assay, Binding Assay, Concentration Assay, Incubation, In Vitro, Kinase Assay, SDS Page, Autoradiography

18) Product Images from "Nuclear Export Signal-Interacting Protein Forms Complexes with Lamin A/C-Nups To Mediate the CRM1-Independent Nuclear Export of Large Hepatitis Delta Antigen"

Article Title: Nuclear Export Signal-Interacting Protein Forms Complexes with Lamin A/C-Nups To Mediate the CRM1-Independent Nuclear Export of Large Hepatitis Delta Antigen

Journal: Journal of Virology

doi: 10.1128/JVI.02357-12

Identification of lamin A/C as an NESI-interacting protein. (A) Coimmunoprecipitation of cellular factors with the NESI-V5His protein. Cell lysates prepared from Huh7 cells transiently transfected with pcDNA-NESI-V5HisTopo encoding an NESI protein with a V5His tag (NESI-V5His) were subjected to a coimmunoprecipitation assay with the anti-V5 antibody and the control preimmune serum. Silver staining is shown. The stars mark the bands of cellular protein 1 and 2 that were coimmunoprecipitated with the NESI-V5His protein. Molecular mass markers are indicated on the left. (B) Coimmunoprecipitation of lamin A/C with NESI-V5His. Cell lysates prepared from Huh7 cells transiently transfected with pcDNA-NESI-V5HisTopo (NESI-V5His) or with pcDNA3.1 vector (Control) were subjected to a coimmunoprecipitation (IP) assay with anti-V5 antibodies and Western blot analysis with anti-lamin A/C (top) and anti-His tag (bottom) antibodies. (C and D) GST pulldown assays with GST-NESI fusion proteins and Huh7 cell lysates. A GST proteins consisting of truncated NESI proteins (indicated at the top by amino acid regions) bound to glutathione-Sepharose 4B beads were incubated independently with Huh7 cell lysates. Following the GST pulldown reaction, Western blot analysis was performed with antibodies specific to lamin A/C (top) and GST (bottom).
Figure Legend Snippet: Identification of lamin A/C as an NESI-interacting protein. (A) Coimmunoprecipitation of cellular factors with the NESI-V5His protein. Cell lysates prepared from Huh7 cells transiently transfected with pcDNA-NESI-V5HisTopo encoding an NESI protein with a V5His tag (NESI-V5His) were subjected to a coimmunoprecipitation assay with the anti-V5 antibody and the control preimmune serum. Silver staining is shown. The stars mark the bands of cellular protein 1 and 2 that were coimmunoprecipitated with the NESI-V5His protein. Molecular mass markers are indicated on the left. (B) Coimmunoprecipitation of lamin A/C with NESI-V5His. Cell lysates prepared from Huh7 cells transiently transfected with pcDNA-NESI-V5HisTopo (NESI-V5His) or with pcDNA3.1 vector (Control) were subjected to a coimmunoprecipitation (IP) assay with anti-V5 antibodies and Western blot analysis with anti-lamin A/C (top) and anti-His tag (bottom) antibodies. (C and D) GST pulldown assays with GST-NESI fusion proteins and Huh7 cell lysates. A GST proteins consisting of truncated NESI proteins (indicated at the top by amino acid regions) bound to glutathione-Sepharose 4B beads were incubated independently with Huh7 cell lysates. Following the GST pulldown reaction, Western blot analysis was performed with antibodies specific to lamin A/C (top) and GST (bottom).

Techniques Used: Transfection, Co-Immunoprecipitation Assay, Silver Staining, Plasmid Preparation, Western Blot, Incubation

Interactions of HDAg-L with NESI, lamin A/C, and nucleoporins. (A) GST pulldown assay. Various GST-NESI fusion proteins and a GST control protein bound to glutathione-Sepharose 4B beads were incubated with the cell lysates prepared from Huh7 cells transiently expressing HDAg-L. Following the GST pulldown reaction, Western blot analysis was performed with antibodies specific to HDAg (top) and GST (bottom). (B) Pro-205 is critical for HDAg-L to interact with NESI. GST pulldown assays were performed with GST-NESI(310–467) and total lysates prepared from Huh7 cells expressing wild-type (WT) HDAg-L or its mutants, HDAg-L(L199A), HDAg-L(D203A), and HDAg-L(P205A), as indicated, followed by Western blotting with antibodies against HDAg-L (top) and GST (bottom). (C to E) Association of HDAg-L with lamin A/C and nucleoporins. Huh7 cells were transfected with plasmids pECE-d-BE, pECE-d-BE(P205A), and pECE-d-SM encoding HDAg-L, HDAg-L(P205A), and HDAg-S, respectively, as indicated. At 2 days posttransfection, cells were harvested and subjected to immunoprecipitation with antibodies specific to lamin A/C (C and D) and nucleoporins (E), followed by Western blotting with antibodies against lamin A/C, nucleoporins, and HDAgs. The star marks the signal of the light chain.
Figure Legend Snippet: Interactions of HDAg-L with NESI, lamin A/C, and nucleoporins. (A) GST pulldown assay. Various GST-NESI fusion proteins and a GST control protein bound to glutathione-Sepharose 4B beads were incubated with the cell lysates prepared from Huh7 cells transiently expressing HDAg-L. Following the GST pulldown reaction, Western blot analysis was performed with antibodies specific to HDAg (top) and GST (bottom). (B) Pro-205 is critical for HDAg-L to interact with NESI. GST pulldown assays were performed with GST-NESI(310–467) and total lysates prepared from Huh7 cells expressing wild-type (WT) HDAg-L or its mutants, HDAg-L(L199A), HDAg-L(D203A), and HDAg-L(P205A), as indicated, followed by Western blotting with antibodies against HDAg-L (top) and GST (bottom). (C to E) Association of HDAg-L with lamin A/C and nucleoporins. Huh7 cells were transfected with plasmids pECE-d-BE, pECE-d-BE(P205A), and pECE-d-SM encoding HDAg-L, HDAg-L(P205A), and HDAg-S, respectively, as indicated. At 2 days posttransfection, cells were harvested and subjected to immunoprecipitation with antibodies specific to lamin A/C (C and D) and nucleoporins (E), followed by Western blotting with antibodies against lamin A/C, nucleoporins, and HDAgs. The star marks the signal of the light chain.

Techniques Used: GST Pulldown Assay, Incubation, Expressing, Western Blot, Transfection, Immunoprecipitation

19) Product Images from "Helicase SUV3, Polynucleotide Phosphorylase, and Mitochondrial Polyadenylation Polymerase Form a Transient Complex to Modulate Mitochondrial mRNA Polyadenylated Tail Lengths in Response to Energetic Changes *"

Article Title: Helicase SUV3, Polynucleotide Phosphorylase, and Mitochondrial Polyadenylation Polymerase Form a Transient Complex to Modulate Mitochondrial mRNA Polyadenylated Tail Lengths in Response to Energetic Changes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.536540

SUV3 serves as a bridge for PNPase and mtPAP binding. A, in vitro binding assay. GST-mtPAP full-length ( FL ) or GST proteins were bound to glutathione-Sepharose 4B beads. Group C was preincubated with increasing amounts of His-SUV3 for 1 h. Subsequently, Groups A, B, and C were incubated with increasing amounts of His-PNPase, whereas Group D was incubated with increasing amounts of preformed SUV3·PNPase complex for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. B , in vitro binding assay. Increasing amounts of un-tagged SUV3 was preincubated with His-PNPase on Ni + resin for 1 h. Subsequently, equal amounts of GST-mtPAP were added to the mixture and further incubated for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. C, size exclusion chromatography (Superdex 200) elution profiles of the purified recombinant SUV3·PNPase·mtPAP complex and the individual proteins. The molecular masses ( M.M .) of the individual proteins were determined by analytical ultracentrifuge. The molecular mass of the SUV3·PNPase·mtPAP complex was approximated from its peak elution volume. D, SDS-PAGE followed by silver staining of the complex. E, schematic representation of the SUV3·PNPase·mtPAP complex.
Figure Legend Snippet: SUV3 serves as a bridge for PNPase and mtPAP binding. A, in vitro binding assay. GST-mtPAP full-length ( FL ) or GST proteins were bound to glutathione-Sepharose 4B beads. Group C was preincubated with increasing amounts of His-SUV3 for 1 h. Subsequently, Groups A, B, and C were incubated with increasing amounts of His-PNPase, whereas Group D was incubated with increasing amounts of preformed SUV3·PNPase complex for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. B , in vitro binding assay. Increasing amounts of un-tagged SUV3 was preincubated with His-PNPase on Ni + resin for 1 h. Subsequently, equal amounts of GST-mtPAP were added to the mixture and further incubated for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. C, size exclusion chromatography (Superdex 200) elution profiles of the purified recombinant SUV3·PNPase·mtPAP complex and the individual proteins. The molecular masses ( M.M .) of the individual proteins were determined by analytical ultracentrifuge. The molecular mass of the SUV3·PNPase·mtPAP complex was approximated from its peak elution volume. D, SDS-PAGE followed by silver staining of the complex. E, schematic representation of the SUV3·PNPase·mtPAP complex.

Techniques Used: Binding Assay, In Vitro, Incubation, SDS Page, Size-exclusion Chromatography, Purification, Recombinant, Silver Staining

N-terminal region of SUV3 binds to the C-terminal region of mtPAP. A and C, schematics of SUV3 constructs used in the in vitro binding assays. B and D, in vitro binding assay. Top panels, immunoblotting of His-tagged mtPAP remained bound to GST-SUV3 fusion proteins on glutathione-Sepharose 4B beads after binding and washing. Bottom panels , protein quantifications of GST-SUV3 fusion proteins by Coomassie Blue staining. *, desired protein species. E and G, schematics of mtPAP constructs used for the in vitro binding assays. F and H, in vitro binding assay. Top panels : immunoblotting of His-tagged SUV3 remained bound to GST-mtPAP fusion proteins on-Sepharose 4B beads after binding and washing. Bottom panels , protein quantifications of GST-mtPAP fusion proteins by Coomassie Blue staining. FL, full-length.
Figure Legend Snippet: N-terminal region of SUV3 binds to the C-terminal region of mtPAP. A and C, schematics of SUV3 constructs used in the in vitro binding assays. B and D, in vitro binding assay. Top panels, immunoblotting of His-tagged mtPAP remained bound to GST-SUV3 fusion proteins on glutathione-Sepharose 4B beads after binding and washing. Bottom panels , protein quantifications of GST-SUV3 fusion proteins by Coomassie Blue staining. *, desired protein species. E and G, schematics of mtPAP constructs used for the in vitro binding assays. F and H, in vitro binding assay. Top panels : immunoblotting of His-tagged SUV3 remained bound to GST-mtPAP fusion proteins on-Sepharose 4B beads after binding and washing. Bottom panels , protein quantifications of GST-mtPAP fusion proteins by Coomassie Blue staining. FL, full-length.

Techniques Used: Construct, In Vitro, Binding Assay, Staining

20) Product Images from "Pleiotrophin signals increased tyrosine phosphorylation of ?-catenin through inactivation of the intrinsic catalytic activity of the receptor-type protein tyrosine phosphatase ?/?"

Article Title: Pleiotrophin signals increased tyrosine phosphorylation of ?-catenin through inactivation of the intrinsic catalytic activity of the receptor-type protein tyrosine phosphatase ?/?

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

doi:

Association of RPTP β/ζ with PTN. ( A ) Lysates of U373-MG glioblastoma cells were immunoprecipitated with anti-RPTP β/ζ monoclonal antibodies. The immunoprecipitates were separated on 6% acrylamide gel, transferred to a poly(vinylidene difluoride) membrane, and probed with anti-RPTP β/ζ antibodies. The arrowheads indicate the RPTP β/ζ spliced products of ≈230, 130, and 85 kDa. ( B ) Western analysis of RPTP β/ζ captured by PTN-Fc. Lysates of U373-MG cells were incubated with PTN-Fc and proteins interactive with PTN-Fc (right lane) were captured with protein A Sepharose-4B beads for 2 h. The beads were washed in cold lysis buffer, boiled in SDS/PAGE sample buffer, and the eluted proteins were separated on an 8% acrylamide gel and analyzed by Western blots probed with anti-RPTP β/ζ monoclonal antibodies. As a control, PTN-Fc was replaced with an equal amount of human IgG (left lane). The arrowheads indicate the ≈130- and ≈85-kDa spliced products of RPTP β/ζ. ( C ) Western analysis of RPTP β/ζ captured by endogenous PTN. Lysates of U373-MG cells were incubated with anti-PTN monoclonal antibodies (right lane) and the complexes were captured with protein A Sepharose-4B beads for 2 h. The beads were washed in cold lysis buffer and boiled in SDS/PAGE sample buffer, and the eluted proteins were separated on an 8% acrylamide gel and analyzed by Western blots probed with anti-RPTP β/ζ monoclonal antibodies. As a control, mouse IgG replaced the anti-PTN antibody (left lane). The arrowheads indicate the ≈130- and ≈85-kDa spliced products of RPTP β/ζ.
Figure Legend Snippet: Association of RPTP β/ζ with PTN. ( A ) Lysates of U373-MG glioblastoma cells were immunoprecipitated with anti-RPTP β/ζ monoclonal antibodies. The immunoprecipitates were separated on 6% acrylamide gel, transferred to a poly(vinylidene difluoride) membrane, and probed with anti-RPTP β/ζ antibodies. The arrowheads indicate the RPTP β/ζ spliced products of ≈230, 130, and 85 kDa. ( B ) Western analysis of RPTP β/ζ captured by PTN-Fc. Lysates of U373-MG cells were incubated with PTN-Fc and proteins interactive with PTN-Fc (right lane) were captured with protein A Sepharose-4B beads for 2 h. The beads were washed in cold lysis buffer, boiled in SDS/PAGE sample buffer, and the eluted proteins were separated on an 8% acrylamide gel and analyzed by Western blots probed with anti-RPTP β/ζ monoclonal antibodies. As a control, PTN-Fc was replaced with an equal amount of human IgG (left lane). The arrowheads indicate the ≈130- and ≈85-kDa spliced products of RPTP β/ζ. ( C ) Western analysis of RPTP β/ζ captured by endogenous PTN. Lysates of U373-MG cells were incubated with anti-PTN monoclonal antibodies (right lane) and the complexes were captured with protein A Sepharose-4B beads for 2 h. The beads were washed in cold lysis buffer and boiled in SDS/PAGE sample buffer, and the eluted proteins were separated on an 8% acrylamide gel and analyzed by Western blots probed with anti-RPTP β/ζ monoclonal antibodies. As a control, mouse IgG replaced the anti-PTN antibody (left lane). The arrowheads indicate the ≈130- and ≈85-kDa spliced products of RPTP β/ζ.

Techniques Used: Immunoprecipitation, Acrylamide Gel Assay, Western Blot, Incubation, Lysis, SDS Page

Physical and functional association of β-catenin with PTN/RPTP β/ζ. ( A ). PTN-Fc is in complex with RPTP β/ζ and β-catenin. PTN-Fc-treated confluent U373-MG cells from 60-mm dish were chemically cross-linked with 3,3′-dithiobis(sulfosuccinimidyl propionate). Lysates from PTN-Fc-treated, chemically cross-linked cells (lanes 1) or Fc- (alone) treated (control) U373-MG cells (lanes 2) were incubated with protein A Sepharose, washed, eluted with SDS sample buffer with 5% 2-mercaptoethanol, and analyzed in 6% SDS gels and Western blots. Lysates from untreated U373-MG cells alone (lanes 3) were also analyzed as a control. Western blots were analyzed with anti-β-catenin ( Right ) or anti-RPTP β/ζ antibodies ( Left ). Arrowheads identify RPTP β/ζ spliced products of ≈250, 230, 130, and 85 kDa ( Left ) and β-catenin (94 kDa) ( Right ). ( B ) β-Catenin interacts with proximal (catalytic) domain of RPTP β/ζ. The GST-D1-RPTP β/ζ wild-type, GST-D1-Cys-1925 → Ser (inactivating) mutant fusion protein or GST alone were expressed and immobilized with glutathione-Sepharose-4B beads, incubated with U373-MG cell lysates, washed, and analyzed in Western analysis with the α-phosphotyrosine antibodies and visualized with the enhanced chemiluminescence ECL-PLUS system ( Lower ). The same blot was reprobed with α-β-catenin antibodies and detected as above ( Upper ).
Figure Legend Snippet: Physical and functional association of β-catenin with PTN/RPTP β/ζ. ( A ). PTN-Fc is in complex with RPTP β/ζ and β-catenin. PTN-Fc-treated confluent U373-MG cells from 60-mm dish were chemically cross-linked with 3,3′-dithiobis(sulfosuccinimidyl propionate). Lysates from PTN-Fc-treated, chemically cross-linked cells (lanes 1) or Fc- (alone) treated (control) U373-MG cells (lanes 2) were incubated with protein A Sepharose, washed, eluted with SDS sample buffer with 5% 2-mercaptoethanol, and analyzed in 6% SDS gels and Western blots. Lysates from untreated U373-MG cells alone (lanes 3) were also analyzed as a control. Western blots were analyzed with anti-β-catenin ( Right ) or anti-RPTP β/ζ antibodies ( Left ). Arrowheads identify RPTP β/ζ spliced products of ≈250, 230, 130, and 85 kDa ( Left ) and β-catenin (94 kDa) ( Right ). ( B ) β-Catenin interacts with proximal (catalytic) domain of RPTP β/ζ. The GST-D1-RPTP β/ζ wild-type, GST-D1-Cys-1925 → Ser (inactivating) mutant fusion protein or GST alone were expressed and immobilized with glutathione-Sepharose-4B beads, incubated with U373-MG cell lysates, washed, and analyzed in Western analysis with the α-phosphotyrosine antibodies and visualized with the enhanced chemiluminescence ECL-PLUS system ( Lower ). The same blot was reprobed with α-β-catenin antibodies and detected as above ( Upper ).

Techniques Used: Functional Assay, Incubation, Western Blot, Mutagenesis

21) Product Images from "Nuclear import of prototype foamy virus transactivator Bel1 is mediated by KPNA1, KPNA6 and KPNA7"

Article Title: Nuclear import of prototype foamy virus transactivator Bel1 is mediated by KPNA1, KPNA6 and KPNA7

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2016.2635

Interaction between 215–221 and KPNAs. 293T cells were transfected with pC3-EGFP-X-GST empty vector or the Bel1 truncated mutant 215–221, as for 215–221 in Fig. 1 , along with plasmids encoding Flag-KPNAs; 48 h post-transfection, cell lysates were incubated with Glutathione Sepharose 4B beads for 4 h at 4°C. Samples from both cell lysates and Glutathione Sepharose 4B beads were subjected to western blot analaysis and probed with anti-Flag, anti-GST and anti-GAPDH antibodies. Representative results from 3 independent experiments are shown.
Figure Legend Snippet: Interaction between 215–221 and KPNAs. 293T cells were transfected with pC3-EGFP-X-GST empty vector or the Bel1 truncated mutant 215–221, as for 215–221 in Fig. 1 , along with plasmids encoding Flag-KPNAs; 48 h post-transfection, cell lysates were incubated with Glutathione Sepharose 4B beads for 4 h at 4°C. Samples from both cell lysates and Glutathione Sepharose 4B beads were subjected to western blot analaysis and probed with anti-Flag, anti-GST and anti-GAPDH antibodies. Representative results from 3 independent experiments are shown.

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

Interaction between Bel1 and KPNAs or KPNB1. 293T cells were transfected with pC3-EGFP-X-GST empty vector or pC3-EGFP-Bel1-GST plasmid along with plasmids that encode Flag-KPNAs or Flag-KPNB1; 48 h post-transfection, cell lysates were incubated with Glutathione Sepharose 4B beads for 4 h at 4°C. Samples from both cell lysates and Glutathione Sepharose 4B beads were subjected to western blot analysis and probed with anti-Flag, anti-GST and anti-GAPDH antibodies. Representative results from 3 independent experiments are shown.
Figure Legend Snippet: Interaction between Bel1 and KPNAs or KPNB1. 293T cells were transfected with pC3-EGFP-X-GST empty vector or pC3-EGFP-Bel1-GST plasmid along with plasmids that encode Flag-KPNAs or Flag-KPNB1; 48 h post-transfection, cell lysates were incubated with Glutathione Sepharose 4B beads for 4 h at 4°C. Samples from both cell lysates and Glutathione Sepharose 4B beads were subjected to western blot analysis and probed with anti-Flag, anti-GST and anti-GAPDH antibodies. Representative results from 3 independent experiments are shown.

Techniques Used: Transfection, Plasmid Preparation, Incubation, Western Blot

22) Product Images from "Opposing effects of polyglutamine expansion on native protein complexes contribute to SCA1"

Article Title: Opposing effects of polyglutamine expansion on native protein complexes contribute to SCA1

Journal: Nature

doi: 10.1038/nature06731

ATXN1-S776D but not ATXN1-S776A interacts with RBM17 (a) Schematic representation of the ATXN1 constructs. (b) RBM17 specifically interacted with ATXN1-S776D in the Y2H screen. AD (activation domain) and DB (DNA binding domain) of Gal4 were fused to human ATXN1 or RBM17, respectively. Y2H controls are: lane 1, negative control; lane 2, weak positive control; and lanes 3–5, strong positive controls. (c) ATXN1 interacted with RBM17 in HEK293T cells by co-AP assays. Top panel shows expression of myc-RBM17 after affinity purification on Glutathione-Sepharose 4B beads, demonstrating the ATXN1/RBM17 interaction (arrow). GST-empty vector was used as a control (−). IB, Immunoblot. (d) Co-IP of Atxn1 with RBM17 from wild-type mouse cerebellar extracts. The anti-RBM17 antibody co-immunoprecipitated Atxn1 (arrow), but not the long and short isoforms of Capicua, CIC-L and CIC-S, respectively (bracket).
Figure Legend Snippet: ATXN1-S776D but not ATXN1-S776A interacts with RBM17 (a) Schematic representation of the ATXN1 constructs. (b) RBM17 specifically interacted with ATXN1-S776D in the Y2H screen. AD (activation domain) and DB (DNA binding domain) of Gal4 were fused to human ATXN1 or RBM17, respectively. Y2H controls are: lane 1, negative control; lane 2, weak positive control; and lanes 3–5, strong positive controls. (c) ATXN1 interacted with RBM17 in HEK293T cells by co-AP assays. Top panel shows expression of myc-RBM17 after affinity purification on Glutathione-Sepharose 4B beads, demonstrating the ATXN1/RBM17 interaction (arrow). GST-empty vector was used as a control (−). IB, Immunoblot. (d) Co-IP of Atxn1 with RBM17 from wild-type mouse cerebellar extracts. The anti-RBM17 antibody co-immunoprecipitated Atxn1 (arrow), but not the long and short isoforms of Capicua, CIC-L and CIC-S, respectively (bracket).

Techniques Used: Construct, Activation Assay, Binding Assay, Negative Control, Positive Control, Expressing, Affinity Purification, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunoprecipitation

23) Product Images from "Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide"

Article Title: Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077288

Identification of CIN85 and CD2AP as LOX-PP interacting proteins in breast cancer cells. ( A ) Extracts from ZR-75 cells transfected with vectors expressing GST or LOX-PP-GST were precipitated with Glutathione-Sepharose 4B beads, resolved by SDS-PAGE and silver stained. The band(s) at ∼85 kDa was analyzed by LC-MS/MS mass spectrometry and identified as CIN85 and CD2AP. *, non-specific proteins. The positions of the co-precipitated CD2AP/CIN85 and LOX-PP proteins are indicated by the solid and large hatched arrows, respectively, and of GST by the dashed line. ( B–C ) GST or LOX-PP-GST (PP-GST) was co-expressed with GFP-CIN85 WT (B) or FLAG-CD2AP (C) in HEK293T cells, and LOX-PP associated proteins isolated by GST-pull down assays and subjected to WB for GFP (B) or FLAG (C) and GST. Input, 4% of lysates (4%). ( D ) Recombinant LOX-PP-myc-His (0.5 µM) was subjected to a GST-pull down assay using 0.5 µM of either GST or GST (G)-CIN85, and WB for the His or CIN85 (Calbiochem) antibody. Input, 5%. ( E ) Samples of whole cell extracts (10 µg) of the indicated human and mouse cells were subjected to WB for CIN85 (Upstate). ( F ) (Left) TX-100 extracts of Hs578T (Upper) or ZR-75 (Lower) cells were immunoprecipitated with mouse IgG or CIN85 (Upstate) antibody, and analyzed for CIN85 (Upstate) and LOX-PP. (Right) TX-100 extracts of Hs578T (upper) or ZR-75 (lower) cells were immunoprecipitated with rabbit IgG or LOX-PP antibodies, and subjected to WB.
Figure Legend Snippet: Identification of CIN85 and CD2AP as LOX-PP interacting proteins in breast cancer cells. ( A ) Extracts from ZR-75 cells transfected with vectors expressing GST or LOX-PP-GST were precipitated with Glutathione-Sepharose 4B beads, resolved by SDS-PAGE and silver stained. The band(s) at ∼85 kDa was analyzed by LC-MS/MS mass spectrometry and identified as CIN85 and CD2AP. *, non-specific proteins. The positions of the co-precipitated CD2AP/CIN85 and LOX-PP proteins are indicated by the solid and large hatched arrows, respectively, and of GST by the dashed line. ( B–C ) GST or LOX-PP-GST (PP-GST) was co-expressed with GFP-CIN85 WT (B) or FLAG-CD2AP (C) in HEK293T cells, and LOX-PP associated proteins isolated by GST-pull down assays and subjected to WB for GFP (B) or FLAG (C) and GST. Input, 4% of lysates (4%). ( D ) Recombinant LOX-PP-myc-His (0.5 µM) was subjected to a GST-pull down assay using 0.5 µM of either GST or GST (G)-CIN85, and WB for the His or CIN85 (Calbiochem) antibody. Input, 5%. ( E ) Samples of whole cell extracts (10 µg) of the indicated human and mouse cells were subjected to WB for CIN85 (Upstate). ( F ) (Left) TX-100 extracts of Hs578T (Upper) or ZR-75 (Lower) cells were immunoprecipitated with mouse IgG or CIN85 (Upstate) antibody, and analyzed for CIN85 (Upstate) and LOX-PP. (Right) TX-100 extracts of Hs578T (upper) or ZR-75 (lower) cells were immunoprecipitated with rabbit IgG or LOX-PP antibodies, and subjected to WB.

Techniques Used: Transfection, Expressing, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation, Western Blot, Recombinant, Pull Down Assay, Immunoprecipitation

24) Product Images from "Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation"

Article Title: Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation

Journal: Nature Communications

doi: 10.1038/s41467-018-07025-z

CUL4A DET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f HEK293T cells were transfected with indicated constructs. The lysates were collected and blotted with anti-Flag and anti-Myc antibody. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
Figure Legend Snippet: CUL4A DET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f HEK293T cells were transfected with indicated constructs. The lysates were collected and blotted with anti-Flag and anti-Myc antibody. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Techniques Used: Immunoprecipitation, Transfection, Construct, Recombinant, Purification, Incubation, Western Blot

25) Product Images from "Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation"

Article Title: Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation

Journal: Nature Communications

doi: 10.1038/s41467-018-07025-z

CUL4A DET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f
Figure Legend Snippet: CUL4A DET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f

Techniques Used: Immunoprecipitation, Transfection, Construct, Recombinant, Purification, Incubation, Western Blot

26) Product Images from "SPARC Regulates Transforming Growth Factor Beta Induced (TGFBI) Extracellular Matrix Deposition and Paclitaxel Response in Ovarian Cancer Cells"

Article Title: SPARC Regulates Transforming Growth Factor Beta Induced (TGFBI) Extracellular Matrix Deposition and Paclitaxel Response in Ovarian Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162698

SPARC directly interacts with TGFBI via its carboxy-terminus. (a) In vitro GST-binding assays. Coomassie stained gel of purified GST and GST-SPARC (aa 18–303) expressed in bacteria. Western blot analysis was performed following GST pull-down from SKOV3 lysates, probed with antibodies specific to the indicated proteins. (b) Coomassie stained gel of bacterially expressed and purified GST, GST-SPARC (aa 18–303), GST-SPARC Nterm (aa 18–134), and GST-SPARC Cterm (aa 154–303) fusion proteins. Western blot analysis was performed following GST pull-down assay from SKOV3 lysates, probed with antibodies specific to the indicated proteins. (c) In vitro binding of purified GST-SPARC to bacterially expressed and purified recombinant TGFBI. GST, GST-SPARC (aa 18–303), or GST-SPARC Nterm (aa 18–134) fusion proteins were incubated with rTGFBI or fibronectin, followed by pull-down with Glutathione sepharose 4B beads. Subsequently, Western blot analysis was performed with antibodies specific to the indicated proteins. Coomassie stained gel represents experimental input.
Figure Legend Snippet: SPARC directly interacts with TGFBI via its carboxy-terminus. (a) In vitro GST-binding assays. Coomassie stained gel of purified GST and GST-SPARC (aa 18–303) expressed in bacteria. Western blot analysis was performed following GST pull-down from SKOV3 lysates, probed with antibodies specific to the indicated proteins. (b) Coomassie stained gel of bacterially expressed and purified GST, GST-SPARC (aa 18–303), GST-SPARC Nterm (aa 18–134), and GST-SPARC Cterm (aa 154–303) fusion proteins. Western blot analysis was performed following GST pull-down assay from SKOV3 lysates, probed with antibodies specific to the indicated proteins. (c) In vitro binding of purified GST-SPARC to bacterially expressed and purified recombinant TGFBI. GST, GST-SPARC (aa 18–303), or GST-SPARC Nterm (aa 18–134) fusion proteins were incubated with rTGFBI or fibronectin, followed by pull-down with Glutathione sepharose 4B beads. Subsequently, Western blot analysis was performed with antibodies specific to the indicated proteins. Coomassie stained gel represents experimental input.

Techniques Used: In Vitro, Binding Assay, Staining, Purification, Western Blot, Pull Down Assay, Recombinant, Incubation

27) Product Images from "SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing"

Article Title: SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing

Journal: EMBO Molecular Medicine

doi: 10.1002/emmm.201100140

Recombinant hSASP14 directly cleaves recombinant filaggrin in vitro Production and purification of hSASP14 by autoprocessing of GST-hSASP28. Purified GST-hSASP28 (arrow) was incubated for the indicated times (0′, 0 min; 60′, 60 min) with 700 mM NaCl at pH 6.0. GST-hSASP28 underwent autoprocessing and produced hSASP14 (arrowhead). Cleaved GST-fusion proteins were removed by passing through Glutathione Sepharose 4B beads to purify hSASP14 (arrowhead). Asterisk indicates a dimer of hSASP14. Cleavage of profilaggrin linker peptide by hSASP14 in vitro . The purified MBP-hFilaggrin/MBP-hFilaggrin-ΔC (arrow) was incubated with or without the purified hSASP14 (arrowhead) with 700 mM NaCl at pH 6.0 for 60 min at 37°C. The linker peptide of profilaggrin between MBP and hFilaggrin in MBP-hFilaggrin/MBP-hFilaggrin-ΔC was cleaved by hSASP14, resulting in the production of MBP (42 kDa), hFilaggrin (37 kDa), and hFilaggrin-ΔC (23 kDa). The N-terminal amino acid sequencing of hFilaggrin (37 kDa) and hFilaggrin-ΔC (23 kDa) protein identified QVSTH amino acids, which corresponded to the linker peptide of profilaggrin. Schematic representation of the mode of processing of MBP-hFilaggrin by hSASP14 as described in B. Schematic representation of the possible cleavage site of profilaggrin by hSASP14. Homodimerized hSASP14 proteins were suggested to primarily cleave between GSFLY-QVSTH in the profilaggrin linker sequence (arrow).
Figure Legend Snippet: Recombinant hSASP14 directly cleaves recombinant filaggrin in vitro Production and purification of hSASP14 by autoprocessing of GST-hSASP28. Purified GST-hSASP28 (arrow) was incubated for the indicated times (0′, 0 min; 60′, 60 min) with 700 mM NaCl at pH 6.0. GST-hSASP28 underwent autoprocessing and produced hSASP14 (arrowhead). Cleaved GST-fusion proteins were removed by passing through Glutathione Sepharose 4B beads to purify hSASP14 (arrowhead). Asterisk indicates a dimer of hSASP14. Cleavage of profilaggrin linker peptide by hSASP14 in vitro . The purified MBP-hFilaggrin/MBP-hFilaggrin-ΔC (arrow) was incubated with or without the purified hSASP14 (arrowhead) with 700 mM NaCl at pH 6.0 for 60 min at 37°C. The linker peptide of profilaggrin between MBP and hFilaggrin in MBP-hFilaggrin/MBP-hFilaggrin-ΔC was cleaved by hSASP14, resulting in the production of MBP (42 kDa), hFilaggrin (37 kDa), and hFilaggrin-ΔC (23 kDa). The N-terminal amino acid sequencing of hFilaggrin (37 kDa) and hFilaggrin-ΔC (23 kDa) protein identified QVSTH amino acids, which corresponded to the linker peptide of profilaggrin. Schematic representation of the mode of processing of MBP-hFilaggrin by hSASP14 as described in B. Schematic representation of the possible cleavage site of profilaggrin by hSASP14. Homodimerized hSASP14 proteins were suggested to primarily cleave between GSFLY-QVSTH in the profilaggrin linker sequence (arrow).

Techniques Used: Recombinant, In Vitro, Purification, Incubation, Produced, Sequencing

28) Product Images from "CRMP5 Interacts with Tubulin to Inhibit Neurite Outgrowth, Thereby Modulating the Function of CRMP2"

Article Title: CRMP5 Interacts with Tubulin to Inhibit Neurite Outgrowth, Thereby Modulating the Function of CRMP2

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0059-10.2010

CRMP5 interacts with MAP2 and tubulin. A , Brain extract from P8 mice was immunoprecipitated by anti-MAP2 antibody, and Western blots were probed for CRMP5 and α-tubulin immunoreactivity. CRMP5 and tubulin were coimmunoprecipitated in a complex with MAP2 protein. Immunoprecipitation of MAP2 is shown as positive control. Note the absence of reactivity in the control IgG. B , Endogenous and overexpressed CRMP5 colocalized with tubulin in NGF-induced differentiated PC12 cells. Left panel, Cells transfected with FLAG-CRMP5 and immunostained with anti-α-tubulin and anti-FLAG antibodies show colocalization of CRMP5 and tubulin in the cytosol and in thin extensions. The inset is a magnification of this area. Middle panel, High magnification of one PC12 cell clearly shows colocalization of CRMP5 and tubulin. Right panel, Endogenous CRMP5 colocalized with tubulin at the cytosolic level and in thin extensions. The inset is a magnification of one PC12 cell. Scale bars, 20 μm. C , Immunoprecipitation of brain extract from P8 mice with anti-α- or anti-β-tubulin antibodies. Western blots were probed for CRMP5 immunoreactivity. CRMP5 was immunoprecipitated in a complex with α- and β-tubulin. As expected, α- and β-tubulin were also coimmunoprecipitated with anti-β- and anti-α-tubulin antibodies, respectively. Control IgG failed to precipitate any protein. D , Overlay assay using purified recombinant CRMP5 (15 μg) subjected to SDS-PAGE and transferred to nitrocellulose membrane. After an overnight incubation with brain extract from P8 mouse (+) or buffer (−), the membrane was probed for MAP2, α-tubulin, or βIII-tubulin immunoreactivity. CRMP5 interacts with MAP2, α-tubulin, or βIII-tubulin present in the brain extract. The intensity of interaction is higher for MAP2 and α-tubulin than for βIII-tubulin. E , Pull down of tubulin from mouse brain extract with GST or GST-CRMP5 (10 μg) conjugated to glutathione Sepharose 4B beads. Western blots of the pellet were probed for tubulin and MAP2 immunoreactivity. GST-CRMP5, but not GST, precipitated MAP2 and α-tubulin. Each result is representative of three to five separate experiments with similar results.
Figure Legend Snippet: CRMP5 interacts with MAP2 and tubulin. A , Brain extract from P8 mice was immunoprecipitated by anti-MAP2 antibody, and Western blots were probed for CRMP5 and α-tubulin immunoreactivity. CRMP5 and tubulin were coimmunoprecipitated in a complex with MAP2 protein. Immunoprecipitation of MAP2 is shown as positive control. Note the absence of reactivity in the control IgG. B , Endogenous and overexpressed CRMP5 colocalized with tubulin in NGF-induced differentiated PC12 cells. Left panel, Cells transfected with FLAG-CRMP5 and immunostained with anti-α-tubulin and anti-FLAG antibodies show colocalization of CRMP5 and tubulin in the cytosol and in thin extensions. The inset is a magnification of this area. Middle panel, High magnification of one PC12 cell clearly shows colocalization of CRMP5 and tubulin. Right panel, Endogenous CRMP5 colocalized with tubulin at the cytosolic level and in thin extensions. The inset is a magnification of one PC12 cell. Scale bars, 20 μm. C , Immunoprecipitation of brain extract from P8 mice with anti-α- or anti-β-tubulin antibodies. Western blots were probed for CRMP5 immunoreactivity. CRMP5 was immunoprecipitated in a complex with α- and β-tubulin. As expected, α- and β-tubulin were also coimmunoprecipitated with anti-β- and anti-α-tubulin antibodies, respectively. Control IgG failed to precipitate any protein. D , Overlay assay using purified recombinant CRMP5 (15 μg) subjected to SDS-PAGE and transferred to nitrocellulose membrane. After an overnight incubation with brain extract from P8 mouse (+) or buffer (−), the membrane was probed for MAP2, α-tubulin, or βIII-tubulin immunoreactivity. CRMP5 interacts with MAP2, α-tubulin, or βIII-tubulin present in the brain extract. The intensity of interaction is higher for MAP2 and α-tubulin than for βIII-tubulin. E , Pull down of tubulin from mouse brain extract with GST or GST-CRMP5 (10 μg) conjugated to glutathione Sepharose 4B beads. Western blots of the pellet were probed for tubulin and MAP2 immunoreactivity. GST-CRMP5, but not GST, precipitated MAP2 and α-tubulin. Each result is representative of three to five separate experiments with similar results.

Techniques Used: Mouse Assay, Immunoprecipitation, Western Blot, Positive Control, Transfection, Overlay Assay, Purification, Recombinant, SDS Page, Incubation

29) Product Images from "Phactr3/Scapinin, a Member of Protein Phosphatase 1 and Actin Regulator (Phactr) Family, Interacts with the Plasma Membrane via Basic and Hydrophobic Residues in the N-Terminus"

Article Title: Phactr3/Scapinin, a Member of Protein Phosphatase 1 and Actin Regulator (Phactr) Family, Interacts with the Plasma Membrane via Basic and Hydrophobic Residues in the N-Terminus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113289

The interaction between the Nt and lipid bilayers. (A) Liposome co-sedimentation assay. Nt was produced as a fusion protein with glutathione transferase (GST-Nt) in the BL21 strain of Escherichia coli and purified with glutathione-Sepharose 4B beads. GST was used as a control. Diagram showing the liposome sedimentation assay. GST and and GST-Nt were centrifuged before use to sediment insoluble proteins and mixed with liposomes (input). After centrifugation, the liposome-bound (ppt) and liposome-unbound (sup) fractions were recovered. Aliquots of each fraction were separated on an SDS-PAGE and stained with Coomassie brilliant blue. (B) Lipid binding assay using the spot array method. Lipid-spotted membrane strips were incubated with GST or GST-Nt and the lipid-bound proteins were detected using anti-GST antibody. LPA, lysophosphatidic acid; PI, phosphatidylinositol; PI(3)P, phosphatidylinositol-(3)-phosphate; PI(4)P, phosphatidylinositol-(4)-phosphate; PI(5), phosphatidylinositol-(5)-phosphate; PE, phosphatidylethanolamine, PC, phosphatidylcholine; SIP, sphingosine-1-phosphate; PI(3,4)P 2 ; phosphatidylinositol-(3,4)-bisphosphate; PI(3,5)P 2 , phosphatidylinositol-(3,5)-bisphosphate; PI(3,4,5)P 3 , phosphatidylinositol-(3,4,5)-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.
Figure Legend Snippet: The interaction between the Nt and lipid bilayers. (A) Liposome co-sedimentation assay. Nt was produced as a fusion protein with glutathione transferase (GST-Nt) in the BL21 strain of Escherichia coli and purified with glutathione-Sepharose 4B beads. GST was used as a control. Diagram showing the liposome sedimentation assay. GST and and GST-Nt were centrifuged before use to sediment insoluble proteins and mixed with liposomes (input). After centrifugation, the liposome-bound (ppt) and liposome-unbound (sup) fractions were recovered. Aliquots of each fraction were separated on an SDS-PAGE and stained with Coomassie brilliant blue. (B) Lipid binding assay using the spot array method. Lipid-spotted membrane strips were incubated with GST or GST-Nt and the lipid-bound proteins were detected using anti-GST antibody. LPA, lysophosphatidic acid; PI, phosphatidylinositol; PI(3)P, phosphatidylinositol-(3)-phosphate; PI(4)P, phosphatidylinositol-(4)-phosphate; PI(5), phosphatidylinositol-(5)-phosphate; PE, phosphatidylethanolamine, PC, phosphatidylcholine; SIP, sphingosine-1-phosphate; PI(3,4)P 2 ; phosphatidylinositol-(3,4)-bisphosphate; PI(3,5)P 2 , phosphatidylinositol-(3,5)-bisphosphate; PI(3,4,5)P 3 , phosphatidylinositol-(3,4,5)-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.

Techniques Used: Sedimentation, Produced, Purification, Centrifugation, SDS Page, Staining, Binding Assay, Incubation

30) Product Images from "The Arabidopsis Cysteine-Rich Receptor-Like Kinase CRK36 Regulates Immunity through Interaction with the Cytoplasmic Kinase BIK1"

Article Title: The Arabidopsis Cysteine-Rich Receptor-Like Kinase CRK36 Regulates Immunity through Interaction with the Cytoplasmic Kinase BIK1

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.01856

Physical interaction between CRK36 and BIK1. (A) Yeast two-hybrid assay for interaction of CRK36 with PTI components. Cytoplasmic kinase domains of FLS2, CERK1, BAK1, and CRK36 and the full-length of BIK1 were fused with GAL4 BD or AD as indicated. Their interactions were tested on selective medium lacking adenine, histidine, leucine and tryptophan (–AHLT) in the absence or presence of X-α-Gal. (B) In vitro GST pull-down assay for interaction between CRK36 and BIK1. GST or GST-fused BIK1 was incubated with the His-tagged kinase domain of CRK36 and precipitated with glutathione sepharose 4B beads. Proteins were detected by immunoblotting with anti-GST and anti-His antibodies. Input shows 1% of the amount used in binding reactions. (C) In vivo co-immunoprecipitation assay for interaction between CRK36 and BIK1. Protein extracts were prepared from N. benthamiana leaves expressing full-lengths of CRK36-HA and BIK1-Myc, and subjected to immunoprecipitation with anti-Myc antibody. Proteins were detected by immunoblotting with anti-HA and anti-Myc antibodies. Input shows 1% of the amount used in the binding reactions. IP, immunoprecipitation. (D) BiFC assay for interaction of CRK36 with BIK1 and FLS2 in Arabidopsis protoplasts. YFP N , YFP C , and their fusions with bZIP63, CRK36, FLS2, and BIK1 were co-expressed in Arabidopsis protoplasts for 24 h, and then treated with 100 nM flg22 for 15 min as indicated. Reconstituted YFP fluorescence was visualized under a confocal microscope. BF, bright field. Bars, 10 μm. (E) Quantitative analysis of interaction of CRK36 with BIK1 and FLS2 in (D) . YFP fluorescence was quantified based on pixel intensity. Values are means ± SD ( n = 12). Asterisk indicates significant difference between untreated and flg22-treated BIK1-YFP n +CRK36-YFP c ( t -test; * P
Figure Legend Snippet: Physical interaction between CRK36 and BIK1. (A) Yeast two-hybrid assay for interaction of CRK36 with PTI components. Cytoplasmic kinase domains of FLS2, CERK1, BAK1, and CRK36 and the full-length of BIK1 were fused with GAL4 BD or AD as indicated. Their interactions were tested on selective medium lacking adenine, histidine, leucine and tryptophan (–AHLT) in the absence or presence of X-α-Gal. (B) In vitro GST pull-down assay for interaction between CRK36 and BIK1. GST or GST-fused BIK1 was incubated with the His-tagged kinase domain of CRK36 and precipitated with glutathione sepharose 4B beads. Proteins were detected by immunoblotting with anti-GST and anti-His antibodies. Input shows 1% of the amount used in binding reactions. (C) In vivo co-immunoprecipitation assay for interaction between CRK36 and BIK1. Protein extracts were prepared from N. benthamiana leaves expressing full-lengths of CRK36-HA and BIK1-Myc, and subjected to immunoprecipitation with anti-Myc antibody. Proteins were detected by immunoblotting with anti-HA and anti-Myc antibodies. Input shows 1% of the amount used in the binding reactions. IP, immunoprecipitation. (D) BiFC assay for interaction of CRK36 with BIK1 and FLS2 in Arabidopsis protoplasts. YFP N , YFP C , and their fusions with bZIP63, CRK36, FLS2, and BIK1 were co-expressed in Arabidopsis protoplasts for 24 h, and then treated with 100 nM flg22 for 15 min as indicated. Reconstituted YFP fluorescence was visualized under a confocal microscope. BF, bright field. Bars, 10 μm. (E) Quantitative analysis of interaction of CRK36 with BIK1 and FLS2 in (D) . YFP fluorescence was quantified based on pixel intensity. Values are means ± SD ( n = 12). Asterisk indicates significant difference between untreated and flg22-treated BIK1-YFP n +CRK36-YFP c ( t -test; * P

Techniques Used: Y2H Assay, In Vitro, Pull Down Assay, Incubation, Binding Assay, In Vivo, Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Bimolecular Fluorescence Complementation Assay, Fluorescence, Microscopy

31) Product Images from "Regulation of N-WASP and the Arp2/3 Complex by Abp1 Controls Neuronal Morphology"

Article Title: Regulation of N-WASP and the Arp2/3 Complex by Abp1 Controls Neuronal Morphology

Journal: PLoS ONE

doi: 10.1371/journal.pone.0000400

Full-length Abp1 and its SH3 domain alone both stimulate actin polymerization in vitro. GST-fusion proteins attached to glutathione sepharose 4B beads were incubated with high speed supernatants of brain extracts supplemented with an energy regenerating mix and Alexa Fluor® 568 G-actin. Actin polymerization is detected on the surface of beads coated with N-WASP PWA (A), full-length Abp1 (B, C) and the Abp1 SH3 domain (D). No polymerization was observed when the beads were coated with a mutated version of the Abp1 SH3 domain (E). Left panels (A–E), actin fluorescence of Alexa Fluor® 568 G-actin; right panels (A′–E′), phase contrast. Bars (A and D) = 10 µm; bars (B, C and E) = 25 µm.
Figure Legend Snippet: Full-length Abp1 and its SH3 domain alone both stimulate actin polymerization in vitro. GST-fusion proteins attached to glutathione sepharose 4B beads were incubated with high speed supernatants of brain extracts supplemented with an energy regenerating mix and Alexa Fluor® 568 G-actin. Actin polymerization is detected on the surface of beads coated with N-WASP PWA (A), full-length Abp1 (B, C) and the Abp1 SH3 domain (D). No polymerization was observed when the beads were coated with a mutated version of the Abp1 SH3 domain (E). Left panels (A–E), actin fluorescence of Alexa Fluor® 568 G-actin; right panels (A′–E′), phase contrast. Bars (A and D) = 10 µm; bars (B, C and E) = 25 µm.

Techniques Used: In Vitro, Incubation, Fluorescence

32) Product Images from "Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions"

Article Title: Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004518

The gp41 cytoplasmic tail binds directly to retromer. A) Native coimmunoprecipitation with anti-CD8 identifies retromer components Vps26 and Vps35 as interacting with the HIV-1 gp41CT. Cell lysates prepared from untransfected (UT), CD8-CIMPR or CD8-gp41CT expressing HeLa cells were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. B) GST-pulldown confirms direct binding of the gp41CT to retromer. Purified recombinant FLAG-tagged retromer complex (3xFLAG-Vps26-Vps29-Vps35-His 6 ) was incubated with purified GST or GST fusion proteins containing the CIMPR (GST-CIMPR) or Envgp41 (GST-gp41CT) cytoplasmic tail and proteins were pulled down with glutathione-Sepharose 4B beads. Bound retromer components Vps26 and Vps35 were detected by immunoblotting.
Figure Legend Snippet: The gp41 cytoplasmic tail binds directly to retromer. A) Native coimmunoprecipitation with anti-CD8 identifies retromer components Vps26 and Vps35 as interacting with the HIV-1 gp41CT. Cell lysates prepared from untransfected (UT), CD8-CIMPR or CD8-gp41CT expressing HeLa cells were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. B) GST-pulldown confirms direct binding of the gp41CT to retromer. Purified recombinant FLAG-tagged retromer complex (3xFLAG-Vps26-Vps29-Vps35-His 6 ) was incubated with purified GST or GST fusion proteins containing the CIMPR (GST-CIMPR) or Envgp41 (GST-gp41CT) cytoplasmic tail and proteins were pulled down with glutathione-Sepharose 4B beads. Bound retromer components Vps26 and Vps35 were detected by immunoblotting.

Techniques Used: Expressing, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Negative Control, Positive Control, Binding Assay, Purification, Recombinant

33) Product Images from "The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein"

Article Title: The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138936

Identification of the domains responsible for the interaction between HSP90 and PABPN1. (A) Representation of full-length HSP90 and the structural domains used to determine the PABPN1 binding domain. (B) Identification of HSP90 domains involved in the PABPN1 interaction. Lysates prepared from HEK293 cells transfected with GFP-tagged A17-PABPN1 and various Flag-tagged HSP90 domain constructs were subjected to IP with an anti-Flag antibody followed by anti-GFP immunoblotting. (C) Representation of PABPN1 and the structural domains used to determine the HSP90 binding domain. (D) Identification of PABPN1 domains involved in the HSP90 interaction. Bacterial GST-HSP90(233–439) fusion proteins, immobilized on glutathione-Sepharose 4B beads, were incubated with lysates from HEK293 cells expressing various HA-tagged PABPN1 domain constructs. The precipitated proteins were subjected to anti-HA immunoblotting. A representative result from three independent experiments is shown. Asterisks highlight the various GST fusion proteins as confirmed by the comparison with the migration of molecular weight marker. IP, immunoprecipitation; PD, pull-down; NB, non-specific band.
Figure Legend Snippet: Identification of the domains responsible for the interaction between HSP90 and PABPN1. (A) Representation of full-length HSP90 and the structural domains used to determine the PABPN1 binding domain. (B) Identification of HSP90 domains involved in the PABPN1 interaction. Lysates prepared from HEK293 cells transfected with GFP-tagged A17-PABPN1 and various Flag-tagged HSP90 domain constructs were subjected to IP with an anti-Flag antibody followed by anti-GFP immunoblotting. (C) Representation of PABPN1 and the structural domains used to determine the HSP90 binding domain. (D) Identification of PABPN1 domains involved in the HSP90 interaction. Bacterial GST-HSP90(233–439) fusion proteins, immobilized on glutathione-Sepharose 4B beads, were incubated with lysates from HEK293 cells expressing various HA-tagged PABPN1 domain constructs. The precipitated proteins were subjected to anti-HA immunoblotting. A representative result from three independent experiments is shown. Asterisks highlight the various GST fusion proteins as confirmed by the comparison with the migration of molecular weight marker. IP, immunoprecipitation; PD, pull-down; NB, non-specific band.

Techniques Used: Binding Assay, Transfection, Construct, Incubation, Expressing, Migration, Molecular Weight, Marker, Immunoprecipitation

34) Product Images from "Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis"

Article Title: Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0078129

Proteomic analysis of amelogenin-interacting proteins in osteoblastic cells. Purified GST-rM180 immobilized on glutathione-Sepharose 4B beads was incubated with no extract (GST-rM180), fractionated soluble protein extract (GST-rM180 + cytoplasm) ( A ) or membrane-associated protein extract (GST-rM180 + membrane) ( B ) prepared from SaOS-2 cells. GST control gels for the both extracts ware also shown to exclude the possibility to non-specific bindings (GST + cytoplasm, GST + membrane). To minimize binding of nonspecific proteins, the cell extracts were pre-cleaned with glutathione beads. The proteins bound to affinity matrices were eluted and separated by isoelectric focusing and SDS-PAGE was performed on a 7.5–15% gradient gel. A typical two-dimensional gel is illustrated. The pH gradient of the separation in the first dimension is shown on the top of the gels, and the molecular weight markers are shown in kDa on the left of the gels. Proteins were visualized with Coomassie brilliant blue staining, excised, trypsinized, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis as described in Table 2 , 3. Magnified views of indicated areas were shown to demonstrate the analyzed spots of amelogenin-interacting proteins (Protein spots).
Figure Legend Snippet: Proteomic analysis of amelogenin-interacting proteins in osteoblastic cells. Purified GST-rM180 immobilized on glutathione-Sepharose 4B beads was incubated with no extract (GST-rM180), fractionated soluble protein extract (GST-rM180 + cytoplasm) ( A ) or membrane-associated protein extract (GST-rM180 + membrane) ( B ) prepared from SaOS-2 cells. GST control gels for the both extracts ware also shown to exclude the possibility to non-specific bindings (GST + cytoplasm, GST + membrane). To minimize binding of nonspecific proteins, the cell extracts were pre-cleaned with glutathione beads. The proteins bound to affinity matrices were eluted and separated by isoelectric focusing and SDS-PAGE was performed on a 7.5–15% gradient gel. A typical two-dimensional gel is illustrated. The pH gradient of the separation in the first dimension is shown on the top of the gels, and the molecular weight markers are shown in kDa on the left of the gels. Proteins were visualized with Coomassie brilliant blue staining, excised, trypsinized, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis as described in Table 2 , 3. Magnified views of indicated areas were shown to demonstrate the analyzed spots of amelogenin-interacting proteins (Protein spots).

Techniques Used: Purification, Incubation, Binding Assay, SDS Page, Two-Dimensional Gel Electrophoresis, Molecular Weight, Staining, Mass Spectrometry

35) Product Images from "Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq"

Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq

Journal: Cell Research

doi: 10.1038/cr.2017.112

Osr1 interacts directly with H2A.Z in vivo and in vitro . (A) Osr1 co-immunoprecipitates with H2A.Z but not H2A from cell extracts. Whole-cell extracts were prepared from NIH3T3 cells co-transfected with Osr1-FLAG-GFPC or Tinf2-FLAG-GFPC and GFPN-HA-H2A or GFPN-HA-H2A.Z for 48 h, followed by immunoprecipitation using anti-FLAG or anti-HA antibodies. The immunoprecipitates were washed with buffers containing 300 or 500 mM NaCl and detected by western blotting with anti-HA antibody. (B) Cartoons showing the full length and truncated H2A.Z proteins expressed in bacteria as MBP fusion proteins, which were purified using Amylose beads. The helices and M6 acid patch domains are indicated. The full length and different truncated Osr1 proteins were expressed in bacteria as GST fusion proteins and purified using glutathione-Sepharose 4B beads. (C) GST or GST-Osr1 fusion proteins bound to beads were incubated with different MBP-H2A.Z fusion proteins. The H2A.Z proteins captured by the beads were detected by western blotting using anti-HA antibody. (D) MBP or MBP-H2A.Z fusion proteins bound to beads were incubated with different GST-Osr1 fusion proteins. The Osr1 proteins captured by the beads were detected by western blotting using anti-FLAG antibody.
Figure Legend Snippet: Osr1 interacts directly with H2A.Z in vivo and in vitro . (A) Osr1 co-immunoprecipitates with H2A.Z but not H2A from cell extracts. Whole-cell extracts were prepared from NIH3T3 cells co-transfected with Osr1-FLAG-GFPC or Tinf2-FLAG-GFPC and GFPN-HA-H2A or GFPN-HA-H2A.Z for 48 h, followed by immunoprecipitation using anti-FLAG or anti-HA antibodies. The immunoprecipitates were washed with buffers containing 300 or 500 mM NaCl and detected by western blotting with anti-HA antibody. (B) Cartoons showing the full length and truncated H2A.Z proteins expressed in bacteria as MBP fusion proteins, which were purified using Amylose beads. The helices and M6 acid patch domains are indicated. The full length and different truncated Osr1 proteins were expressed in bacteria as GST fusion proteins and purified using glutathione-Sepharose 4B beads. (C) GST or GST-Osr1 fusion proteins bound to beads were incubated with different MBP-H2A.Z fusion proteins. The H2A.Z proteins captured by the beads were detected by western blotting using anti-HA antibody. (D) MBP or MBP-H2A.Z fusion proteins bound to beads were incubated with different GST-Osr1 fusion proteins. The Osr1 proteins captured by the beads were detected by western blotting using anti-FLAG antibody.

Techniques Used: In Vivo, In Vitro, Transfection, Immunoprecipitation, Western Blot, Purification, Incubation

36) Product Images from "AP2/ERF Family Transcription Factors ORA59 and RAP2.3 Interact in the Nucleus and Function Together in Ethylene Responses"

Article Title: AP2/ERF Family Transcription Factors ORA59 and RAP2.3 Interact in the Nucleus and Function Together in Ethylene Responses

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.01675

Physical interaction of ORA59 with RAP2.3. (A) Yeast two-hybrid assay. ORA59 with N-terminal 60 amino acids deleted (ORA59Δ1-60) and full-length RAP2.3 were fused with GAL4 AD and BD, respectively. Their interactions were tested on selective media SD/-AHLT and in the presence of X-α-Gal. (B) In vitro GST pull-down assay. GST or ORA59-GST was incubated with RAP2.3-His and precipitated with glutathione sepharose 4B beads. Proteins were detected by immunoblotting with anti-GST and anti-His antibodies. Input shows 1% of the amount used in binding reactions. WB, western blotting. (C) BiFC assay. YFP NE , YFP CE , and their fusion proteins bZIP63 NE , bZIP63 CE , ORA59 NE , and RAP2.3 CE were expressed in Arabidopsis protoplasts as indicated. YFP fluorescence signals were visualized under a confocal microscope. Bars, 10 μm. Experiments were repeated three times with similar results.
Figure Legend Snippet: Physical interaction of ORA59 with RAP2.3. (A) Yeast two-hybrid assay. ORA59 with N-terminal 60 amino acids deleted (ORA59Δ1-60) and full-length RAP2.3 were fused with GAL4 AD and BD, respectively. Their interactions were tested on selective media SD/-AHLT and in the presence of X-α-Gal. (B) In vitro GST pull-down assay. GST or ORA59-GST was incubated with RAP2.3-His and precipitated with glutathione sepharose 4B beads. Proteins were detected by immunoblotting with anti-GST and anti-His antibodies. Input shows 1% of the amount used in binding reactions. WB, western blotting. (C) BiFC assay. YFP NE , YFP CE , and their fusion proteins bZIP63 NE , bZIP63 CE , ORA59 NE , and RAP2.3 CE were expressed in Arabidopsis protoplasts as indicated. YFP fluorescence signals were visualized under a confocal microscope. Bars, 10 μm. Experiments were repeated three times with similar results.

Techniques Used: Y2H Assay, In Vitro, Pull Down Assay, Incubation, Binding Assay, Western Blot, Bimolecular Fluorescence Complementation Assay, Fluorescence, Microscopy

37) Product Images from "AP2/ERF Family Transcription Factors ORA59 and RAP2.3 Interact in the Nucleus and Function Together in Ethylene Responses"

Article Title: AP2/ERF Family Transcription Factors ORA59 and RAP2.3 Interact in the Nucleus and Function Together in Ethylene Responses

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.01675

Physical interaction of ORA59 with RAP2.3. (A) Yeast two-hybrid assay. ORA59 with N-terminal 60 amino acids deleted (ORA59Δ1-60) and full-length RAP2.3 were fused with GAL4 AD and BD, respectively. Their interactions were tested on selective media SD/-AHLT and in the presence of X-α-Gal. (B) In vitro GST pull-down assay. GST or ORA59-GST was incubated with RAP2.3-His and precipitated with glutathione sepharose 4B beads. Proteins were detected by immunoblotting with anti-GST and anti-His antibodies. Input shows 1% of the amount used in binding reactions. WB, western blotting. (C) BiFC assay. YFP NE , YFP CE , and their fusion proteins bZIP63 NE , bZIP63 CE , ORA59 NE , and RAP2.3 CE were expressed in Arabidopsis protoplasts as indicated. YFP fluorescence signals were visualized under a confocal microscope. Bars, 10 μm. Experiments were repeated three times with similar results.
Figure Legend Snippet: Physical interaction of ORA59 with RAP2.3. (A) Yeast two-hybrid assay. ORA59 with N-terminal 60 amino acids deleted (ORA59Δ1-60) and full-length RAP2.3 were fused with GAL4 AD and BD, respectively. Their interactions were tested on selective media SD/-AHLT and in the presence of X-α-Gal. (B) In vitro GST pull-down assay. GST or ORA59-GST was incubated with RAP2.3-His and precipitated with glutathione sepharose 4B beads. Proteins were detected by immunoblotting with anti-GST and anti-His antibodies. Input shows 1% of the amount used in binding reactions. WB, western blotting. (C) BiFC assay. YFP NE , YFP CE , and their fusion proteins bZIP63 NE , bZIP63 CE , ORA59 NE , and RAP2.3 CE were expressed in Arabidopsis protoplasts as indicated. YFP fluorescence signals were visualized under a confocal microscope. Bars, 10 μm. Experiments were repeated three times with similar results.

Techniques Used: Y2H Assay, In Vitro, Pull Down Assay, Incubation, Binding Assay, Western Blot, Bimolecular Fluorescence Complementation Assay, Fluorescence, Microscopy

38) Product Images from "CFTR-associated ligand is a negative regulator of Mrp2 expression"

Article Title: CFTR-associated ligand is a negative regulator of Mrp2 expression

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00100.2016

Mrp2 binds to CAL via the COOH-terminal PDZ-binding motif in GST pull-down assays. A : diagram of GST-rat Mrp2 COOH-terminus constructs for GST pull-down assays. a : Full-length rat Mrp2 consists of three membrane-spanning domains (in green) and an intracellular COOH-terminal tail (amino acid 1255–1541, in yellow). b : Construct 1 encodes GST (in orange) and amino acid 1255–1541 of rMrp2, including the COOH-terminal PDZ-binding motif that is composed of 4 amino acids (red asterisks). c : Construct 2 encodes GST and amino acid 1255–1538 of rMrp2 with deletion of the last 3 amino acids. d : Construct 3 encodes GST and amino acid 1255–1534 of rMrp2 with deletion of the last 7 amino acids. B : GST pull-down assays. HEK-293 cells were transfected with Lipofectamine 2000, and lysates of cells transfected with HA-CAL were incubated with GST control or GST-rMrp2 fusion protein encoded by the constructs shown in A . The samples were then supplemented with glutathione Sepharose 4B beads. Both the pull-down complex ( top ) and the unbound fraction ( bottom ) were immunoblotted with anti-HA antibody. Note the presence of HA-CAL in the pull-down complex when the COOH-terminus PDZ-binding motif of Mrp2 was included in the GST-Mrp2 bait, but was not detected in the pull-down complex when the COOH-terminus PDZ-binding motif was deleted in ( Construct 1 vs. Construct 2 and Construct 3 ). The bottom panel confirms the presence of HA-CAL in the unbound fraction of all of the pull-down assays. Data are representative of three independent experiments.
Figure Legend Snippet: Mrp2 binds to CAL via the COOH-terminal PDZ-binding motif in GST pull-down assays. A : diagram of GST-rat Mrp2 COOH-terminus constructs for GST pull-down assays. a : Full-length rat Mrp2 consists of three membrane-spanning domains (in green) and an intracellular COOH-terminal tail (amino acid 1255–1541, in yellow). b : Construct 1 encodes GST (in orange) and amino acid 1255–1541 of rMrp2, including the COOH-terminal PDZ-binding motif that is composed of 4 amino acids (red asterisks). c : Construct 2 encodes GST and amino acid 1255–1538 of rMrp2 with deletion of the last 3 amino acids. d : Construct 3 encodes GST and amino acid 1255–1534 of rMrp2 with deletion of the last 7 amino acids. B : GST pull-down assays. HEK-293 cells were transfected with Lipofectamine 2000, and lysates of cells transfected with HA-CAL were incubated with GST control or GST-rMrp2 fusion protein encoded by the constructs shown in A . The samples were then supplemented with glutathione Sepharose 4B beads. Both the pull-down complex ( top ) and the unbound fraction ( bottom ) were immunoblotted with anti-HA antibody. Note the presence of HA-CAL in the pull-down complex when the COOH-terminus PDZ-binding motif of Mrp2 was included in the GST-Mrp2 bait, but was not detected in the pull-down complex when the COOH-terminus PDZ-binding motif was deleted in ( Construct 1 vs. Construct 2 and Construct 3 ). The bottom panel confirms the presence of HA-CAL in the unbound fraction of all of the pull-down assays. Data are representative of three independent experiments.

Techniques Used: Binding Assay, Construct, Transfection, Incubation

39) Product Images from "Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1"

Article Title: Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200108089

MURF family members interact with SUMO modifying enzymes ISOT-3 and Ubc9, but only MURF-1 interacts with the transcriptional regulator GMEB-1. (A) Y2H screens using full-length cDNAs of individual MURF family members as baits identified ISOT-3 (light gray) and Ubc9 (black) as MURF-binding proteins. However, GMEB-1 (dark gray) was found to interact only with MURF-1. β-Galactosidase assays were performed to confirm positive clones, and the levels were compared with colonies transformed with each prey construct and the empty bait vector (white). Data are presented as mean levels of β-galactosidase from triplicate experiments ± SD. ***, P > 0.001. (B) RT-PCR analysis of human heart total RNA revealed that GMEB-1 mRNA transcripts are detectable in heart (H) and skeletal (Sk) tissues. Lane 1, no reverse transcriptase control in human heart RNA (−); lane 2, 511-bp GMEB-1 PCR product amplified from human heart RNA (+); lane 3, 511-bp GMEB-1 PCR product amplified from human skeletal RNA (+); lane 4, no reverse transcriptase control in human skeletal RNA (−). (C) GMEB-1 specifically binds to MURF-1 in GST pull-down assays. GMEB-1 was translated in vitro (lane 3). When incubated with bacterially expressed GST–MURF-1 fusion peptides, GMEB-1 and MURF-1 binding to glutathione–sepharose 4B beads was detectable (lane 2). Lane 1 contains no detectable binding of GMEB-1 to the beads alone. IVT, in vitro translated. (D) GMEB-1–GFP targets to the nuclei of cardiac myocytes (a and c). MURF-1 staining also was present in some of the nuclei that contained GMEB-1–GFP (b). Note, MURF-1 is also detected at the M-line region in the same myocytes (b, double arrows). Expression of GMEB-1–GFP in cardiac myocytes does not appear to affect the integrity of the COOH-terminal region of titin (d, staining with anti-titin A168–170 antibodies). Double arrows mark regular, striated titin staining. N, nuclei. Bars, 10 μm.
Figure Legend Snippet: MURF family members interact with SUMO modifying enzymes ISOT-3 and Ubc9, but only MURF-1 interacts with the transcriptional regulator GMEB-1. (A) Y2H screens using full-length cDNAs of individual MURF family members as baits identified ISOT-3 (light gray) and Ubc9 (black) as MURF-binding proteins. However, GMEB-1 (dark gray) was found to interact only with MURF-1. β-Galactosidase assays were performed to confirm positive clones, and the levels were compared with colonies transformed with each prey construct and the empty bait vector (white). Data are presented as mean levels of β-galactosidase from triplicate experiments ± SD. ***, P > 0.001. (B) RT-PCR analysis of human heart total RNA revealed that GMEB-1 mRNA transcripts are detectable in heart (H) and skeletal (Sk) tissues. Lane 1, no reverse transcriptase control in human heart RNA (−); lane 2, 511-bp GMEB-1 PCR product amplified from human heart RNA (+); lane 3, 511-bp GMEB-1 PCR product amplified from human skeletal RNA (+); lane 4, no reverse transcriptase control in human skeletal RNA (−). (C) GMEB-1 specifically binds to MURF-1 in GST pull-down assays. GMEB-1 was translated in vitro (lane 3). When incubated with bacterially expressed GST–MURF-1 fusion peptides, GMEB-1 and MURF-1 binding to glutathione–sepharose 4B beads was detectable (lane 2). Lane 1 contains no detectable binding of GMEB-1 to the beads alone. IVT, in vitro translated. (D) GMEB-1–GFP targets to the nuclei of cardiac myocytes (a and c). MURF-1 staining also was present in some of the nuclei that contained GMEB-1–GFP (b). Note, MURF-1 is also detected at the M-line region in the same myocytes (b, double arrows). Expression of GMEB-1–GFP in cardiac myocytes does not appear to affect the integrity of the COOH-terminal region of titin (d, staining with anti-titin A168–170 antibodies). Double arrows mark regular, striated titin staining. N, nuclei. Bars, 10 μm.

Techniques Used: Binding Assay, Clone Assay, Transformation Assay, Construct, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, In Vitro, Incubation, Staining, Expressing

40) Product Images from "Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis"

Article Title: Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0078129

Proteomic analysis of amelogenin-interacting proteins in osteoblastic cells. Purified GST-rM180 immobilized on glutathione-Sepharose 4B beads was incubated with no extract (GST-rM180), fractionated soluble protein extract (GST-rM180 + cytoplasm) ( A ) or membrane-associated protein extract (GST-rM180 + membrane) ( B ) prepared from SaOS-2 cells. GST control gels for the both extracts ware also shown to exclude the possibility to non-specific bindings (GST + cytoplasm, GST + membrane). To minimize binding of nonspecific proteins, the cell extracts were pre-cleaned with glutathione beads. The proteins bound to affinity matrices were eluted and separated by isoelectric focusing and SDS-PAGE was performed on a 7.5–15% gradient gel. A typical two-dimensional gel is illustrated. The pH gradient of the separation in the first dimension is shown on the top of the gels, and the molecular weight markers are shown in kDa on the left of the gels. Proteins were visualized with Coomassie brilliant blue staining, excised, trypsinized, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis as described in Table 2 , 3. Magnified views of indicated areas were shown to demonstrate the analyzed spots of amelogenin-interacting proteins (Protein spots).
Figure Legend Snippet: Proteomic analysis of amelogenin-interacting proteins in osteoblastic cells. Purified GST-rM180 immobilized on glutathione-Sepharose 4B beads was incubated with no extract (GST-rM180), fractionated soluble protein extract (GST-rM180 + cytoplasm) ( A ) or membrane-associated protein extract (GST-rM180 + membrane) ( B ) prepared from SaOS-2 cells. GST control gels for the both extracts ware also shown to exclude the possibility to non-specific bindings (GST + cytoplasm, GST + membrane). To minimize binding of nonspecific proteins, the cell extracts were pre-cleaned with glutathione beads. The proteins bound to affinity matrices were eluted and separated by isoelectric focusing and SDS-PAGE was performed on a 7.5–15% gradient gel. A typical two-dimensional gel is illustrated. The pH gradient of the separation in the first dimension is shown on the top of the gels, and the molecular weight markers are shown in kDa on the left of the gels. Proteins were visualized with Coomassie brilliant blue staining, excised, trypsinized, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis as described in Table 2 , 3. Magnified views of indicated areas were shown to demonstrate the analyzed spots of amelogenin-interacting proteins (Protein spots).

Techniques Used: Purification, Incubation, Binding Assay, SDS Page, Two-Dimensional Gel Electrophoresis, Molecular Weight, Staining, Mass Spectrometry

41) Product Images from "Entry of Hepatitis B Virus into Immortalized Human Primary Hepatocytes by Clathrin-Dependent Endocytosis"

Article Title: Entry of Hepatitis B Virus into Immortalized Human Primary Hepatocytes by Clathrin-Dependent Endocytosis

Journal: Journal of Virology

doi: 10.1128/JVI.00873-12

Interaction between LHBsAg and CHC or AP-2. (A) (Top) Schematic representation of the amino acid (a.a.) residues within the LHBsAg subdomains, designated pre-S1, pre-S2, and S; (bottom) Western blot results when LHBsAg cDNA fragments coding for amino acids 1 to 111, 111 to 274, or 274 to 389 were cloned into pGEX-6p-1 for expression in E. coli and purification as GST fusion proteins and antibodies against GST were used to detect the expression of the GST fusion proteins. Arrowheads, leaky expression of proteins. (B) GST pulldown assay. GST-LHBsAg fusion proteins or GST bound to glutathione-Sepharose 4B beads were incubated with lysates of HuS-E/2 cells, and then, after GST pulldown, Western blot analysis was performed using antibodies against CHC, AP-1, AP-2, or GST. The positions of molecular mass markers are shown on the left. (C and D) Coimmunoprecipitation and Western blot analysis. HuS-E/2 cells were transfected with plasmid pcDNA3.0-HA-LHBsAg, pcDNA3.0-HA-MHBsAg, or pcDNA3.0-HA-SHBsAg coding, respectively, for HA-tagged LHBsAg, MHBsAg, or SHBsAg, and then, at 2 days posttransfection, the cells were harvested and subjected to immunoprecipitation (IP) with antibodies specific for HA (C) or CHC (D), followed by Western blot analysis with antibodies against HA, AP-2, or CHC, as indicated. NT, nontransfected cells. The molecular mass markers are indicated on the left. Asterisks, proteins coimmunoprecipitated with CHC; arrowheads, nonspecific bands.
Figure Legend Snippet: Interaction between LHBsAg and CHC or AP-2. (A) (Top) Schematic representation of the amino acid (a.a.) residues within the LHBsAg subdomains, designated pre-S1, pre-S2, and S; (bottom) Western blot results when LHBsAg cDNA fragments coding for amino acids 1 to 111, 111 to 274, or 274 to 389 were cloned into pGEX-6p-1 for expression in E. coli and purification as GST fusion proteins and antibodies against GST were used to detect the expression of the GST fusion proteins. Arrowheads, leaky expression of proteins. (B) GST pulldown assay. GST-LHBsAg fusion proteins or GST bound to glutathione-Sepharose 4B beads were incubated with lysates of HuS-E/2 cells, and then, after GST pulldown, Western blot analysis was performed using antibodies against CHC, AP-1, AP-2, or GST. The positions of molecular mass markers are shown on the left. (C and D) Coimmunoprecipitation and Western blot analysis. HuS-E/2 cells were transfected with plasmid pcDNA3.0-HA-LHBsAg, pcDNA3.0-HA-MHBsAg, or pcDNA3.0-HA-SHBsAg coding, respectively, for HA-tagged LHBsAg, MHBsAg, or SHBsAg, and then, at 2 days posttransfection, the cells were harvested and subjected to immunoprecipitation (IP) with antibodies specific for HA (C) or CHC (D), followed by Western blot analysis with antibodies against HA, AP-2, or CHC, as indicated. NT, nontransfected cells. The molecular mass markers are indicated on the left. Asterisks, proteins coimmunoprecipitated with CHC; arrowheads, nonspecific bands.

Techniques Used: Western Blot, Clone Assay, Expressing, Purification, GST Pulldown Assay, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation

42) Product Images from "FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus"

Article Title: FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

Journal: Nature Communications

doi: 10.1038/ncomms10939

SNX27 interacts directly with FAM21 among the WASH complex components. ( a ) HEK293T cells were transfected with 3 × FLAG-tagged-WASH complex proteins. Lysates were incubated with GST or GST-SNX27 proteins and precipitated with Glutathione Sepharose 4B beads. Precipitates were then immunoblotted as shown. ( b ) Schematic representation of FAM21 constructs used. LFa motifs and α-helices are indicated. ( c , d ) HA-SNX27 was co-transfected with the indicated Myc-tagged constructs in HEK293T cells, and lysates were immunoprecipitated with anti-haemagglutinin (HA) antibodies and immunoblotted. Asterisks (*) denote IgG heavy chains. ( e ) GST or GST-SNX27 fusion proteins were incubated with indicated His-tagged FAM21 proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted as indicated. ( f ) Biotinylated peptides corresponding to FAM21 residues 40–79 and 592–600 were incubated with CBP-SNX27 and precipitated using streptavidin-conjugated beads. Precipitates were immunoblotted for CBP.
Figure Legend Snippet: SNX27 interacts directly with FAM21 among the WASH complex components. ( a ) HEK293T cells were transfected with 3 × FLAG-tagged-WASH complex proteins. Lysates were incubated with GST or GST-SNX27 proteins and precipitated with Glutathione Sepharose 4B beads. Precipitates were then immunoblotted as shown. ( b ) Schematic representation of FAM21 constructs used. LFa motifs and α-helices are indicated. ( c , d ) HA-SNX27 was co-transfected with the indicated Myc-tagged constructs in HEK293T cells, and lysates were immunoprecipitated with anti-haemagglutinin (HA) antibodies and immunoblotted. Asterisks (*) denote IgG heavy chains. ( e ) GST or GST-SNX27 fusion proteins were incubated with indicated His-tagged FAM21 proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted as indicated. ( f ) Biotinylated peptides corresponding to FAM21 residues 40–79 and 592–600 were incubated with CBP-SNX27 and precipitated using streptavidin-conjugated beads. Precipitates were immunoblotted for CBP.

Techniques Used: Transfection, Incubation, Construct, Immunoprecipitation

43) Product Images from "DNA Damage Induced MutS Homologue hMSH4 Acetylation"

Article Title: DNA Damage Induced MutS Homologue hMSH4 Acetylation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms141020966

hMSH4 interacts with hMof. ( A ) Recombinant hMof was produced as a glutathione S -transferase-tagged fusion protein and was co-expressed with hMSH4. Soluble cell lysates were used for GST pull-down analysis. Western blot analysis was performed to detect the expression of hMSH4 protein; (B) Negative controls for GST pull-down assay. In the absence of GST-hMof, glutathione-Sepharose 4B beads could not directly pull down hMSH4 even in the presence of GST tag; ( C ) Co-immunoprecipitation analysis of hMSH4 and hMof interaction in human cells. Myc-hMSH4 and Flag-hMof expression in 293T cells was validated by Western blotting. IR treatment was performed 48 h after transfection. The α-Flag antibody was used to perform co-immunoprecipitation analysis, and co-immunoprecipitated hMSH4 was validated by Western blot analysis.
Figure Legend Snippet: hMSH4 interacts with hMof. ( A ) Recombinant hMof was produced as a glutathione S -transferase-tagged fusion protein and was co-expressed with hMSH4. Soluble cell lysates were used for GST pull-down analysis. Western blot analysis was performed to detect the expression of hMSH4 protein; (B) Negative controls for GST pull-down assay. In the absence of GST-hMof, glutathione-Sepharose 4B beads could not directly pull down hMSH4 even in the presence of GST tag; ( C ) Co-immunoprecipitation analysis of hMSH4 and hMof interaction in human cells. Myc-hMSH4 and Flag-hMof expression in 293T cells was validated by Western blotting. IR treatment was performed 48 h after transfection. The α-Flag antibody was used to perform co-immunoprecipitation analysis, and co-immunoprecipitated hMSH4 was validated by Western blot analysis.

Techniques Used: Recombinant, Produced, Western Blot, Expressing, Pull Down Assay, Immunoprecipitation, Transfection

44) Product Images from "PP5 (PPP5C) is a phosphatase of Dvl2"

Article Title: PP5 (PPP5C) is a phosphatase of Dvl2

Journal: Scientific Reports

doi: 10.1038/s41598-018-21124-3

PP5 interacts with Dvl2. ( a ) Transfected HEK293T cells were processed for coimmunoprecipitation with FLAG M2 beads and samples were analysed by WB. ( b ) HEK293T cells were transfected as indicated and processed for Co-IP with GFP-Trap beads. Asterisk indicates nonspecific signal from enriched EYFP-PP5-H304A protein. ( c ) FLAG-Dvl2 purified from transfected HEK293T cells was incubated with PP5 protein purified from E. coli together with Glutathione Sepharose 4B beads. GST was used as a negative control. ( d , e ) HEK293T cells were transfected as indicated and processed for Co-IP with FLAG M2 beads. WT, wild-type PP5. K97A, PP5-K97A. H304A, PP5-H304A. Asterisk indicates nonspecific bands. ( f ) Schematic of PP5 mutants used in ( g ). ( g ) HEK293T cells were transfected as indicated and processed for Co-IP with FLAG M2 beads. Samples were then analysed by WB. ( h ) Schematic of Dvl2 truncations used in ( i ). ( i .
Figure Legend Snippet: PP5 interacts with Dvl2. ( a ) Transfected HEK293T cells were processed for coimmunoprecipitation with FLAG M2 beads and samples were analysed by WB. ( b ) HEK293T cells were transfected as indicated and processed for Co-IP with GFP-Trap beads. Asterisk indicates nonspecific signal from enriched EYFP-PP5-H304A protein. ( c ) FLAG-Dvl2 purified from transfected HEK293T cells was incubated with PP5 protein purified from E. coli together with Glutathione Sepharose 4B beads. GST was used as a negative control. ( d , e ) HEK293T cells were transfected as indicated and processed for Co-IP with FLAG M2 beads. WT, wild-type PP5. K97A, PP5-K97A. H304A, PP5-H304A. Asterisk indicates nonspecific bands. ( f ) Schematic of PP5 mutants used in ( g ). ( g ) HEK293T cells were transfected as indicated and processed for Co-IP with FLAG M2 beads. Samples were then analysed by WB. ( h ) Schematic of Dvl2 truncations used in ( i ). ( i .

Techniques Used: Transfection, Western Blot, Co-Immunoprecipitation Assay, Purification, Incubation, Negative Control

45) Product Images from "Duplication and Diversification of the Spermidine/Spermine N1-acetyltransferase 1 Genes in Zebrafish"

Article Title: Duplication and Diversification of the Spermidine/Spermine N1-acetyltransferase 1 Genes in Zebrafish

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054017

Protein-protein interactions of zebrafish family of Ssat1 proteins. (A) GST, GST-Ssat1a, GST-Ssat1b, and GST-Ssat1c were purified from bacteria. Lysates of cells transfected with the plasmid encoding Ssat1b were incubated with GST (Lane 1) or GST-Ssat1a (Lane 2). Lysates of cells transfected with the plasmid encoding Ssat1c were incubated with GST (Lane 3), GST-Ssat1a (Lane 4), or GST-Ssat1b (Lane 5). (B) GST and GST fused with the cytosolic domain of zebrafish Integrin 9α (GST-Intg9α_c) were purified from bacteria. Lysates of cells transfected with the plasmid encoding Ssat1a (lanes 1 and 2), Ssat1b (lanes 3 and 4), or Ssat1c (lanes 5 and 6) were incubated with GST (lanes 1, 3 and 5) or GST-Intg9α_c (lanes 2, 4 and 6). (C) Lysates of cells transfected with the plasmid encoding the myc-tagged PAS-B domain of Zebrafish Hif-1α was incubated with GST (lane 1), GST-Ssat1a (lane 2), GST-Ssat1b (lane 3), or GST-Ssat1c (lane 4). Bound proteins in each sample were pulled down with glutathione Sepharose 4B beads and analyzed by western blotting.
Figure Legend Snippet: Protein-protein interactions of zebrafish family of Ssat1 proteins. (A) GST, GST-Ssat1a, GST-Ssat1b, and GST-Ssat1c were purified from bacteria. Lysates of cells transfected with the plasmid encoding Ssat1b were incubated with GST (Lane 1) or GST-Ssat1a (Lane 2). Lysates of cells transfected with the plasmid encoding Ssat1c were incubated with GST (Lane 3), GST-Ssat1a (Lane 4), or GST-Ssat1b (Lane 5). (B) GST and GST fused with the cytosolic domain of zebrafish Integrin 9α (GST-Intg9α_c) were purified from bacteria. Lysates of cells transfected with the plasmid encoding Ssat1a (lanes 1 and 2), Ssat1b (lanes 3 and 4), or Ssat1c (lanes 5 and 6) were incubated with GST (lanes 1, 3 and 5) or GST-Intg9α_c (lanes 2, 4 and 6). (C) Lysates of cells transfected with the plasmid encoding the myc-tagged PAS-B domain of Zebrafish Hif-1α was incubated with GST (lane 1), GST-Ssat1a (lane 2), GST-Ssat1b (lane 3), or GST-Ssat1c (lane 4). Bound proteins in each sample were pulled down with glutathione Sepharose 4B beads and analyzed by western blotting.

Techniques Used: Purification, Transfection, Plasmid Preparation, Incubation, Western Blot

46) Product Images from "Glycogen Synthase Kinase-3 promotes cell survival, growth and PAX3 levels in human melanoma cells"

Article Title: Glycogen Synthase Kinase-3 promotes cell survival, growth and PAX3 levels in human melanoma cells

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-11-0387

) corresponding to sites in A. The pGex2T-PAX3PD construct fuses glutathione S-transferase (GST) to the N-terminal end of the paired domain (PD) and contains amino acids 34–161. pGex2T-PAX3PDHD-WT possesses amino acids 34–297 including the PD, octapeptide (O) and the homeodomain (HD). The amino acid sequence of O and the first GSK-3β recognition motif (S/T-X 3 -S/T) is represented (wild-type (WT) sequence shown, amino acids 186–219). The construct pGex2T-PAX3PDHD-ΔSERAS has the entire series of GSK-3β recognition motifs removed (deleted sequence, (−ΔSERAS) sequence shown, with the removal of amino acids 189–211). D, GSK-3β and PAX3 kinase assay. The kinase assay was performed on empty glutathione sepharose-4B beads without (lane-1) or with GSK-3β (lane-2), pGex2T-PAX3PDHD-WT without (lane-3) or with GSK-3β (lane-4), pGex2T-PAX3PD without (lane-5) or with GSK-3β (lane-6) and pGex2T-PAX3PDHD-ΔSERAS minus (lane-7) or plus GSK-3β (lane-8). The top panel displays the kinase assay with an asterisk indicating an auto-phosphorylation band. The bottom panel is a coomassie-stained gel visualizing the input bound to the beads. E–F, tandem mass spectrometry determines Ser205 and Ser197/Ser201 of PAX3 are phosphorylated by GSK-3β in-vitro . Precursor ion masses were measured in the Orbitrap analyzer and MS 2 spectra were acquired in the LTQ mass spectrometer. E, MS 2 spectra of pSer205 (n-formyl) ASAPQSDEGpSDIDSEPDLPLK (MS mass deviation, 12 ppm). F, MS 2 spectra of peptide AS*APQS*DEGSDIDSEPDLPLK phosphorylated at either Ser197 or Ser201 (MS mass deviation, 23 ppm). The presence of ion Y12 at mass 1328.14m/z in the Y-ion series fragmentation of the MS 2 spectra exclude Ser209 and Ser205 at the phosphorylation site, however, there is insufficient MS 2 ion evidence (b1–9) to pinpoint the phosphorylation site specifically to Ser197 or Ser201, thus both sites with an * are potential sites of phosphorylation. Note: peptide is displayed C- to N-terminus due to the predominant Y-ion fragmentation.
Figure Legend Snippet: ) corresponding to sites in A. The pGex2T-PAX3PD construct fuses glutathione S-transferase (GST) to the N-terminal end of the paired domain (PD) and contains amino acids 34–161. pGex2T-PAX3PDHD-WT possesses amino acids 34–297 including the PD, octapeptide (O) and the homeodomain (HD). The amino acid sequence of O and the first GSK-3β recognition motif (S/T-X 3 -S/T) is represented (wild-type (WT) sequence shown, amino acids 186–219). The construct pGex2T-PAX3PDHD-ΔSERAS has the entire series of GSK-3β recognition motifs removed (deleted sequence, (−ΔSERAS) sequence shown, with the removal of amino acids 189–211). D, GSK-3β and PAX3 kinase assay. The kinase assay was performed on empty glutathione sepharose-4B beads without (lane-1) or with GSK-3β (lane-2), pGex2T-PAX3PDHD-WT without (lane-3) or with GSK-3β (lane-4), pGex2T-PAX3PD without (lane-5) or with GSK-3β (lane-6) and pGex2T-PAX3PDHD-ΔSERAS minus (lane-7) or plus GSK-3β (lane-8). The top panel displays the kinase assay with an asterisk indicating an auto-phosphorylation band. The bottom panel is a coomassie-stained gel visualizing the input bound to the beads. E–F, tandem mass spectrometry determines Ser205 and Ser197/Ser201 of PAX3 are phosphorylated by GSK-3β in-vitro . Precursor ion masses were measured in the Orbitrap analyzer and MS 2 spectra were acquired in the LTQ mass spectrometer. E, MS 2 spectra of pSer205 (n-formyl) ASAPQSDEGpSDIDSEPDLPLK (MS mass deviation, 12 ppm). F, MS 2 spectra of peptide AS*APQS*DEGSDIDSEPDLPLK phosphorylated at either Ser197 or Ser201 (MS mass deviation, 23 ppm). The presence of ion Y12 at mass 1328.14m/z in the Y-ion series fragmentation of the MS 2 spectra exclude Ser209 and Ser205 at the phosphorylation site, however, there is insufficient MS 2 ion evidence (b1–9) to pinpoint the phosphorylation site specifically to Ser197 or Ser201, thus both sites with an * are potential sites of phosphorylation. Note: peptide is displayed C- to N-terminus due to the predominant Y-ion fragmentation.

Techniques Used: Construct, Sequencing, Kinase Assay, Staining, Mass Spectrometry, In Vitro

47) Product Images from "Interplay between Polyadenylate-Binding Protein 1 and Kaposi's Sarcoma-Associated Herpesvirus ORF57 in Accumulation of Polyadenylated Nuclear RNA, a Viral Long Noncoding RNA"

Article Title: Interplay between Polyadenylate-Binding Protein 1 and Kaposi's Sarcoma-Associated Herpesvirus ORF57 in Accumulation of Polyadenylated Nuclear RNA, a Viral Long Noncoding RNA

Journal: Journal of Virology

doi: 10.1128/JVI.01693-12

KSHV ORF57 interacts with PABPC1. (A) ORF57 interacts with PABPC1, but not with E1B-AP5, as shown by coimmunoprecipitation assays. Total cell extracts from TREx BCBL1-Rta cells induced by Dox (1 μg/ml) and treated with a mixture of RNase A and T1 were immunoprecipitated with preimmune mouse IgG- or monoclonal anti-ORF57 antibody-coated beads and examined for PABPC1 and E1B-AP5 by Western blotting (left). Conversely, the same extracts treated with a mixture of RNase A and T1 were immunoprecipitated with nonspecific rabbit IgG- or polyclonal anti-PABPC1 or anti-E1B-AP5 antibody-coated beads and blotted for ORF57 by Western blotting (right). (B) PABPC1 directly interacts with ORF57, as shown by GST pulldown assays. Full-length PABPC1 protein with an N-terminal GST tag or untagged GST was immobilized on glutathione-Sepharose 4B beads and incubated at 4°C overnight in IP buffer complemented with KSHV ORF57 protein containing a C-terminal FLAG tag that was expressed from baculovirus. The glutathione-Sepharose 4B beads without any GST protein were used as an additional negative control. After binding, the beads were washed five times with IP buffer, resuspended in 2× SDS sample buffer, and analyzed by Western blotting with two antibodies, an anti-GST antibody recognizing PABPC1 and an anti-FLAG antibody that recognizes the epitope-tagged ORF57.
Figure Legend Snippet: KSHV ORF57 interacts with PABPC1. (A) ORF57 interacts with PABPC1, but not with E1B-AP5, as shown by coimmunoprecipitation assays. Total cell extracts from TREx BCBL1-Rta cells induced by Dox (1 μg/ml) and treated with a mixture of RNase A and T1 were immunoprecipitated with preimmune mouse IgG- or monoclonal anti-ORF57 antibody-coated beads and examined for PABPC1 and E1B-AP5 by Western blotting (left). Conversely, the same extracts treated with a mixture of RNase A and T1 were immunoprecipitated with nonspecific rabbit IgG- or polyclonal anti-PABPC1 or anti-E1B-AP5 antibody-coated beads and blotted for ORF57 by Western blotting (right). (B) PABPC1 directly interacts with ORF57, as shown by GST pulldown assays. Full-length PABPC1 protein with an N-terminal GST tag or untagged GST was immobilized on glutathione-Sepharose 4B beads and incubated at 4°C overnight in IP buffer complemented with KSHV ORF57 protein containing a C-terminal FLAG tag that was expressed from baculovirus. The glutathione-Sepharose 4B beads without any GST protein were used as an additional negative control. After binding, the beads were washed five times with IP buffer, resuspended in 2× SDS sample buffer, and analyzed by Western blotting with two antibodies, an anti-GST antibody recognizing PABPC1 and an anti-FLAG antibody that recognizes the epitope-tagged ORF57.

Techniques Used: Immunoprecipitation, Western Blot, Incubation, FLAG-tag, Negative Control, Binding Assay

48) Product Images from "Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling"

Article Title: Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18010148

Rif activity was inhibited upon mitogenic stimulation mediated by MAPK or PI3K activation. ( A ) Domain structure of mDia1. Abbreviations: G, GTPase binding region necessary for RhoA binding; DID, diaphanous inhibitory domain; DD, dimerization domain; CC, coiled coil; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoinhibitory domain; ( B ) pGEX-4T1-RifWT (1–195 aa) and pGEX-4T1-mDia1-G-DID (73–370 aa) recombinant proteins were generated in an E. coli protein expression system and soluble protein was immobilized to glutathione-Sepharose 4B beads for the use in a Rif activation assay. GST-Rif (1–195 aa) was cleaved by thrombin and then the released Rif was loaded with GDP or GTP. mDia1-G-DID beads were then incubated with GDP- or GTP-loaded Rif at 4 °C for 1 h. The beads were washed three times with lysis buffer and eluted with sample buffer for immunoblotting. HEK293 cell lysate was added to mimic the complex intracellular environment; ( C ) HeLa cells were serum starved overnight, then treated with 100 ng/mL Epidermal Growth Factor (EGF), 8 ng/mL Lysophosphatidic Acid (LPA), 1 µg/mL Sphingosine 1-Phosphate (SIP) or 10% serum for 3 min and Rif activation levels then examined by the Rif activation assay described in ( B ); ( D ) HeLa cells were serum starved overnight, then pretreated with or without p38 MAPK, PI3K, or p42/44 MAPK inhibitors for 30 min and then stimulated with 100 ng/mL EGF for 5 min. Rif activation levels were examined by the Rif activation assay. Rif activation levels were quantified by immunoblotting. Data are means ± S.E.M ( n = 3). * p
Figure Legend Snippet: Rif activity was inhibited upon mitogenic stimulation mediated by MAPK or PI3K activation. ( A ) Domain structure of mDia1. Abbreviations: G, GTPase binding region necessary for RhoA binding; DID, diaphanous inhibitory domain; DD, dimerization domain; CC, coiled coil; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoinhibitory domain; ( B ) pGEX-4T1-RifWT (1–195 aa) and pGEX-4T1-mDia1-G-DID (73–370 aa) recombinant proteins were generated in an E. coli protein expression system and soluble protein was immobilized to glutathione-Sepharose 4B beads for the use in a Rif activation assay. GST-Rif (1–195 aa) was cleaved by thrombin and then the released Rif was loaded with GDP or GTP. mDia1-G-DID beads were then incubated with GDP- or GTP-loaded Rif at 4 °C for 1 h. The beads were washed three times with lysis buffer and eluted with sample buffer for immunoblotting. HEK293 cell lysate was added to mimic the complex intracellular environment; ( C ) HeLa cells were serum starved overnight, then treated with 100 ng/mL Epidermal Growth Factor (EGF), 8 ng/mL Lysophosphatidic Acid (LPA), 1 µg/mL Sphingosine 1-Phosphate (SIP) or 10% serum for 3 min and Rif activation levels then examined by the Rif activation assay described in ( B ); ( D ) HeLa cells were serum starved overnight, then pretreated with or without p38 MAPK, PI3K, or p42/44 MAPK inhibitors for 30 min and then stimulated with 100 ng/mL EGF for 5 min. Rif activation levels were examined by the Rif activation assay. Rif activation levels were quantified by immunoblotting. Data are means ± S.E.M ( n = 3). * p

Techniques Used: Activity Assay, Activation Assay, Binding Assay, Recombinant, Generated, Expressing, Incubation, Lysis

49) Product Images from "OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation 1"

Article Title: OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation 1

Journal: Plant Physiology

doi: 10.1104/pp.15.01668

The TPR domain of BSK3 prevents it from interacting with AtBSU1. A, Yeast two-hybrid assays of the interaction between full-length OsBSK3, the kinase domain of OsBSK3 (N390), and the TPR domain of OsBSK3 (TPR). B, Yeast two-hybrid assays of the interaction between full-length AtBSK3, full-length OsBSK3, the kinase domain of AtBSK3 (N334), and N390 with AtBSU1, AtBIN2, and the TPR domain from OsBSK3. C, AtBSU1 showed increased binding with the kinase domains of OsBSK3 and AtBSK3. The recombinant 6His-tagged kinase domain and full-length versions of OsBSK3 (N390 and OsBSK3) or AtBSK3 (N334 and AtBSK3) were dot blotted onto nitrocellulose membranes and then incubated with MBP-AtBSU1 and horseradish peroxidase-labeled anti-MBP antibodies. Total protein was visualized by Ponceau S staining. D, OsBRI1 phosphorylation prevents the TPR and kinase domains of OsBSK3 from binding. GST-TPR on Glutathione Sepharose 4B beads was used to pull down 6His-tagged N390, which had been incubated with MBP-tagged OsBRI1 kinase domain in the presence (pN390) or absence (N390) of ATP. The proteins were blotted onto nitrocellulose membranes and detected using anti-His or -GST antibodies. E, Ser-215 phosphorylation reduced the binding of the kinase and TPR domains of OsBSK3. Shown are quantitative β-glycosidase activity assays of the interaction between the TPR domain and wild type or Ser-215-substituted mutant form of N390. A one-way ANOVA was performed. Statistically significant differences are indicated by different lowercase letters ( P
Figure Legend Snippet: The TPR domain of BSK3 prevents it from interacting with AtBSU1. A, Yeast two-hybrid assays of the interaction between full-length OsBSK3, the kinase domain of OsBSK3 (N390), and the TPR domain of OsBSK3 (TPR). B, Yeast two-hybrid assays of the interaction between full-length AtBSK3, full-length OsBSK3, the kinase domain of AtBSK3 (N334), and N390 with AtBSU1, AtBIN2, and the TPR domain from OsBSK3. C, AtBSU1 showed increased binding with the kinase domains of OsBSK3 and AtBSK3. The recombinant 6His-tagged kinase domain and full-length versions of OsBSK3 (N390 and OsBSK3) or AtBSK3 (N334 and AtBSK3) were dot blotted onto nitrocellulose membranes and then incubated with MBP-AtBSU1 and horseradish peroxidase-labeled anti-MBP antibodies. Total protein was visualized by Ponceau S staining. D, OsBRI1 phosphorylation prevents the TPR and kinase domains of OsBSK3 from binding. GST-TPR on Glutathione Sepharose 4B beads was used to pull down 6His-tagged N390, which had been incubated with MBP-tagged OsBRI1 kinase domain in the presence (pN390) or absence (N390) of ATP. The proteins were blotted onto nitrocellulose membranes and detected using anti-His or -GST antibodies. E, Ser-215 phosphorylation reduced the binding of the kinase and TPR domains of OsBSK3. Shown are quantitative β-glycosidase activity assays of the interaction between the TPR domain and wild type or Ser-215-substituted mutant form of N390. A one-way ANOVA was performed. Statistically significant differences are indicated by different lowercase letters ( P

Techniques Used: Binding Assay, Recombinant, Incubation, Labeling, Staining, Activity Assay, Mutagenesis

50) Product Images from "The Chaperone Protein SmgGDS Interacts with Small GTPases Entering the Prenylation Pathway by Recognizing the Last Amino Acid in the CAAX Motif *"

Article Title: The Chaperone Protein SmgGDS Interacts with Small GTPases Entering the Prenylation Pathway by Recognizing the Last Amino Acid in the CAAX Motif *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.527192

SmgGDS-607 binds directly and specifically to K-Ras CVIL, and SmgGDS-558 binds unprenylated WT K-Ras CVIM or CVIL in vitro . A , 100 ng of recombinant His-tagged SmgGDS-607 and SmgGDS-558 were allowed to interact with 250 ng of recombinant GST-Myc-tagged full-length K-Ras CVIM or K-Ras CVIL proteins prebound to glutathione-Sepharose 4B beads. The complexes were isolated by pelleting the beads and subjected to ECL-Western blotting using a SmgGDS antibody. The results are shown as the optical density of the SmgGDS protein signal in the immunoblots and are the means ± S.E. of three independent experiments. ns , not significant; **, p
Figure Legend Snippet: SmgGDS-607 binds directly and specifically to K-Ras CVIL, and SmgGDS-558 binds unprenylated WT K-Ras CVIM or CVIL in vitro . A , 100 ng of recombinant His-tagged SmgGDS-607 and SmgGDS-558 were allowed to interact with 250 ng of recombinant GST-Myc-tagged full-length K-Ras CVIM or K-Ras CVIL proteins prebound to glutathione-Sepharose 4B beads. The complexes were isolated by pelleting the beads and subjected to ECL-Western blotting using a SmgGDS antibody. The results are shown as the optical density of the SmgGDS protein signal in the immunoblots and are the means ± S.E. of three independent experiments. ns , not significant; **, p

Techniques Used: In Vitro, Recombinant, Isolation, Western Blot

51) Product Images from "Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking"

Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15022929

Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
Figure Legend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

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

52) Product Images from "Induction of Apoptosis in Human Prostate Cancer Cells by Insulin-Like Growth Factor Binding Protein-3 Does Not Require Binding to Retinoid X Receptor-?"

Article Title: Induction of Apoptosis in Human Prostate Cancer Cells by Insulin-Like Growth Factor Binding Protein-3 Does Not Require Binding to Retinoid X Receptor-?

Journal: Endocrinology

doi: 10.1210/en.2007-1315

Binding of YFP-IGFBP-3 mutant proteins to RXR-α. A, In vitro binding of YFP-IGFBP-3 mutant proteins to GST-RXR-α fusion proteins. GST-RXR-α (expressed in E . coli ) or GST control was coupled to Glutathione-Sepharose-4B beads. PC-3 cells were transfected with YFP empty vector (lane 1), YFP-WT-IGFBP-3 (lane 2), YFP-MDGEA-IGFBP-3 (lane 3), YFP-HBD-6m-IGFBP-3 (lane 4), YFP-HBD-9m-IGFBP-3 (lane 5), and YFP-HBD-11m-IGFBP-3 (lane 6). After 24 h, total cell lysates were prepared and incubated overnight at 4 C with beads that had been coupled with GST-RXR or GST. Bound proteins were eluted and examined by Western blotting using antibodies to GFP, which recognize YFP ( upper-left panel ). Similar results were obtained in two other experiments. Lower-left panel , Lysates used for the GST pull down were blotted with anti-GFP. Anti-GFP binds to approximately 27-kDa YFP ( open arrowhead , lane 1) and to the YFP moiety of the approximately 67-kDa YFP-IGFBP-3 fusion proteins ( solid arrowheads, upper and lower-left panels ). For clarity, lanes separating the individual lanes shown have been deleted. Lanes 1–3 and lanes 4–6 were taken from different gels. The greater intensity of the YFP-HBD-9m-IGFBP-3 lysate signal relative to the YFP-HBD-11m-IGFBP-3 lysate in this immunoblot is not consistently observed, and does not account for the inability to detect YFP-HBD-11m-IGFBP-3 by GST-RXR pull down. As an additional control, the GST-RXR and GST pull-down blots were stripped and immunoblotted with anti-RAR ( upper-right panel ). Decreased binding of RAR to GST-RXR, however, would not be expected in our experiment because of the large excess of GST-RXR. B, Coimmunoprecipitation of YFP-IGFBP-3 mutants and RXR-α from PC-3 cell lysates. PC-3 cells were transfected with pCMX-RXR-α expression vector and either YFP-HBD-11m-IGFBP-3 (lanes 1 and 2) or YFP-HBD-6m-IGFBP-3 (lanes 3 and 4). After 24 h, cell lysates were prepared, precleared, and equal aliquots were incubated with rabbit IgG control (−, lanes 1 and 3) or with antibody to RXR-α (+, lanes 2 and 4). After 16 h, Protein A/G Plus Agarose beads were added to all the samples. After high-stringency washes, the immunoprecipitated proteins were eluted and analyzed by Western blotting using antibody to GFP to identify the YFP-IGFBP-3 mutant proteins. The position of immunoreactive YFP-IGFBP-3 is indicated by a solid arrowhead . The lanes shown are from the same gel. Lanes separating lanes 1 and 2 from lanes 3 and 4 have been removed for clarity of presentation, and the images spliced to put them in closer proximity.
Figure Legend Snippet: Binding of YFP-IGFBP-3 mutant proteins to RXR-α. A, In vitro binding of YFP-IGFBP-3 mutant proteins to GST-RXR-α fusion proteins. GST-RXR-α (expressed in E . coli ) or GST control was coupled to Glutathione-Sepharose-4B beads. PC-3 cells were transfected with YFP empty vector (lane 1), YFP-WT-IGFBP-3 (lane 2), YFP-MDGEA-IGFBP-3 (lane 3), YFP-HBD-6m-IGFBP-3 (lane 4), YFP-HBD-9m-IGFBP-3 (lane 5), and YFP-HBD-11m-IGFBP-3 (lane 6). After 24 h, total cell lysates were prepared and incubated overnight at 4 C with beads that had been coupled with GST-RXR or GST. Bound proteins were eluted and examined by Western blotting using antibodies to GFP, which recognize YFP ( upper-left panel ). Similar results were obtained in two other experiments. Lower-left panel , Lysates used for the GST pull down were blotted with anti-GFP. Anti-GFP binds to approximately 27-kDa YFP ( open arrowhead , lane 1) and to the YFP moiety of the approximately 67-kDa YFP-IGFBP-3 fusion proteins ( solid arrowheads, upper and lower-left panels ). For clarity, lanes separating the individual lanes shown have been deleted. Lanes 1–3 and lanes 4–6 were taken from different gels. The greater intensity of the YFP-HBD-9m-IGFBP-3 lysate signal relative to the YFP-HBD-11m-IGFBP-3 lysate in this immunoblot is not consistently observed, and does not account for the inability to detect YFP-HBD-11m-IGFBP-3 by GST-RXR pull down. As an additional control, the GST-RXR and GST pull-down blots were stripped and immunoblotted with anti-RAR ( upper-right panel ). Decreased binding of RAR to GST-RXR, however, would not be expected in our experiment because of the large excess of GST-RXR. B, Coimmunoprecipitation of YFP-IGFBP-3 mutants and RXR-α from PC-3 cell lysates. PC-3 cells were transfected with pCMX-RXR-α expression vector and either YFP-HBD-11m-IGFBP-3 (lanes 1 and 2) or YFP-HBD-6m-IGFBP-3 (lanes 3 and 4). After 24 h, cell lysates were prepared, precleared, and equal aliquots were incubated with rabbit IgG control (−, lanes 1 and 3) or with antibody to RXR-α (+, lanes 2 and 4). After 16 h, Protein A/G Plus Agarose beads were added to all the samples. After high-stringency washes, the immunoprecipitated proteins were eluted and analyzed by Western blotting using antibody to GFP to identify the YFP-IGFBP-3 mutant proteins. The position of immunoreactive YFP-IGFBP-3 is indicated by a solid arrowhead . The lanes shown are from the same gel. Lanes separating lanes 1 and 2 from lanes 3 and 4 have been removed for clarity of presentation, and the images spliced to put them in closer proximity.

Techniques Used: Binding Assay, Mutagenesis, In Vitro, Transfection, Plasmid Preparation, Incubation, Western Blot, Expressing, Immunoprecipitation

53) Product Images from "The C-Terminal Domain of the Novel Essential Protein Gcp Is Critical for Interaction with Another Essential Protein YeaZ of Staphylococcus aureus"

Article Title: The C-Terminal Domain of the Novel Essential Protein Gcp Is Critical for Interaction with Another Essential Protein YeaZ of Staphylococcus aureus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020163

The determination of Gcp binding to YeaZ. (A) Yeast two hybrid analysis of interaction between Gcp and YeaZ on histidine, leucine, and tryptophan drop out synthetic complete (SC-His, -Leu, -Trp) plates with 3-amino-1,2,4,-triazole (3-AT) and SC plates lacking adenine, leucine, and tryptophan (SC-Ade, -Leu, -Trp). Gcp was fused with the activation domain and YeaZ was fused to binding domain. The minus signs indicate empty vector controls. (B) In vitro immunoprecipitation analysis of interaction between Gcp and YeaZ. His-tagged Gcp and GST-tagged YeaZ were purified from E. coli . Gcp and YeaZ were incubated with Glutathione Sepharose 4B beads together or separately. Western blotting was carried out with rabbit-anti-Gcp serum to detect Gcp. Lane 1: purified Gcp protein was loaded as a positive control.
Figure Legend Snippet: The determination of Gcp binding to YeaZ. (A) Yeast two hybrid analysis of interaction between Gcp and YeaZ on histidine, leucine, and tryptophan drop out synthetic complete (SC-His, -Leu, -Trp) plates with 3-amino-1,2,4,-triazole (3-AT) and SC plates lacking adenine, leucine, and tryptophan (SC-Ade, -Leu, -Trp). Gcp was fused with the activation domain and YeaZ was fused to binding domain. The minus signs indicate empty vector controls. (B) In vitro immunoprecipitation analysis of interaction between Gcp and YeaZ. His-tagged Gcp and GST-tagged YeaZ were purified from E. coli . Gcp and YeaZ were incubated with Glutathione Sepharose 4B beads together or separately. Western blotting was carried out with rabbit-anti-Gcp serum to detect Gcp. Lane 1: purified Gcp protein was loaded as a positive control.

Techniques Used: Binding Assay, Activation Assay, Plasmid Preparation, In Vitro, Immunoprecipitation, Purification, Incubation, Western Blot, Positive Control

54) Product Images from "14-3-3? Protein Regulates Anterograde Transport of the Human ?-Opioid Receptor (hKOPR) *"

Article Title: 14-3-3? Protein Regulates Anterograde Transport of the Human ?-Opioid Receptor (hKOPR) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.359679

A , interaction of the hKOPR C-tail with 14-3-3ζ in rat brain extracts by pulldown assay is shown. Rat brains were homogenized, solubilized, and centrifuged at 100,000 × g for 40 min, and the supernatants were filtered through 0.45-μm and then 0.22-μm membranes. The filtrate was used for pulldown experiments by incubating with glutathione-agarose beads preloaded with GST or GST-hKOPR-C-tail overnight at 4 °C. IB , immunoblot. B , direct interaction between the hKOR C-tail and 14-3-3ζ is shown. Purified 14-3-3ζ was incubated with glutathione-Sepharose 4B beads preloaded with GST and GST-hKOPR C-tail overnight at 4 °C. A and B , the beads were washed extensively, and the bound proteins were eluted from the beads, resolved by 8% SDS-PAGE, and transferred onto Immobilon™-P PVDF membranes. A 1/250 supernatant was also loaded as an input control. Upper panel , 14-3-3 was detected by a rabbit anti-14-3-3ζ antibody. Lower panel , the same membrane was stained with Ponceau S, showing the relative sizes and the amounts of the GST and GST-hKOPR C-tail loaded. The figure represents one of the three experiments with similar results.
Figure Legend Snippet: A , interaction of the hKOPR C-tail with 14-3-3ζ in rat brain extracts by pulldown assay is shown. Rat brains were homogenized, solubilized, and centrifuged at 100,000 × g for 40 min, and the supernatants were filtered through 0.45-μm and then 0.22-μm membranes. The filtrate was used for pulldown experiments by incubating with glutathione-agarose beads preloaded with GST or GST-hKOPR-C-tail overnight at 4 °C. IB , immunoblot. B , direct interaction between the hKOR C-tail and 14-3-3ζ is shown. Purified 14-3-3ζ was incubated with glutathione-Sepharose 4B beads preloaded with GST and GST-hKOPR C-tail overnight at 4 °C. A and B , the beads were washed extensively, and the bound proteins were eluted from the beads, resolved by 8% SDS-PAGE, and transferred onto Immobilon™-P PVDF membranes. A 1/250 supernatant was also loaded as an input control. Upper panel , 14-3-3 was detected by a rabbit anti-14-3-3ζ antibody. Lower panel , the same membrane was stained with Ponceau S, showing the relative sizes and the amounts of the GST and GST-hKOPR C-tail loaded. The figure represents one of the three experiments with similar results.

Techniques Used: Purification, Incubation, SDS Page, Staining

55) Product Images from "Fructose Uptake in Bifidobacterium longum NCC2705 Is Mediated by an ATP-binding Cassette Transporter *"

Article Title: Fructose Uptake in Bifidobacterium longum NCC2705 Is Mediated by an ATP-binding Cassette Transporter *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.266213

SDS-PAGE and GST pulldown assays to analyze protein-protein interactions of the FruEKFG ABC transporter subunits. A , Coomassie-stained SDS-PAGE of crude extracts ( lanes 1 , 3 , 5 , 7 , and 9 ; 30 μg of protein were loaded per sample) and purified proteins ( lanes 2 , 4 , 6 , 8 , and 10 ; 5–10 μg of purified protein was loaded per sample) of IPTG-induced E. coli BL21(DE3) containing pET32a-FruE ( lanes 1 and 2 ), pET32a-FruK ( lanes 3 and 4 ), pGEX-4T-1-FruF ( lanes 5 and 6 ), pGEX-4T-1-FruG ( lanes 7 and 8 ), and pGEX-4T-1-FruE ( lanes 9 and 10 ). Lane M , molecular weight marker. The proteins were purified by Ni + affinity column (His-FruE and His-FruK) or GST beads (GST-FruF, GST-FruG, and GST-FruE). B , GST pulldown assays probing interactions between FruE or FruK with the membrane permeases FruF and FruG. For pulldown, 25 μg of GST fusion protein was incubated with 5 μl of glutathione-Sepharose 4B beads for 2 h in PBS at 4 °C. Then 200 μl of lysate containing a total of 25 μg of protein of an E. coli BL21 strain expressing the respective His 6 -tagged protein were added, and the bound proteins were precipitated by centrifugation. Lane 1 , GST + His-FruE (negative control); lane 2 , GST-FruF + His-FruE; lane 3 , His-FruE (positive control); lane 4 , GST + His-FruK (negative control); lane 5 , GST-FruF + His-FruK; lane 6 , His-FruK (positive control); lane 7 , GST + His-FruE (negative control); lane 8 , GST-FruG + His-FruE; lane 9 , His-FruE (positive control); lane 10 , GST + His-FruK (negative control); lane 11 , GST-FruF + His-FruK; Lane 12 , His-FruK (positive control). IB , immunoblot.
Figure Legend Snippet: SDS-PAGE and GST pulldown assays to analyze protein-protein interactions of the FruEKFG ABC transporter subunits. A , Coomassie-stained SDS-PAGE of crude extracts ( lanes 1 , 3 , 5 , 7 , and 9 ; 30 μg of protein were loaded per sample) and purified proteins ( lanes 2 , 4 , 6 , 8 , and 10 ; 5–10 μg of purified protein was loaded per sample) of IPTG-induced E. coli BL21(DE3) containing pET32a-FruE ( lanes 1 and 2 ), pET32a-FruK ( lanes 3 and 4 ), pGEX-4T-1-FruF ( lanes 5 and 6 ), pGEX-4T-1-FruG ( lanes 7 and 8 ), and pGEX-4T-1-FruE ( lanes 9 and 10 ). Lane M , molecular weight marker. The proteins were purified by Ni + affinity column (His-FruE and His-FruK) or GST beads (GST-FruF, GST-FruG, and GST-FruE). B , GST pulldown assays probing interactions between FruE or FruK with the membrane permeases FruF and FruG. For pulldown, 25 μg of GST fusion protein was incubated with 5 μl of glutathione-Sepharose 4B beads for 2 h in PBS at 4 °C. Then 200 μl of lysate containing a total of 25 μg of protein of an E. coli BL21 strain expressing the respective His 6 -tagged protein were added, and the bound proteins were precipitated by centrifugation. Lane 1 , GST + His-FruE (negative control); lane 2 , GST-FruF + His-FruE; lane 3 , His-FruE (positive control); lane 4 , GST + His-FruK (negative control); lane 5 , GST-FruF + His-FruK; lane 6 , His-FruK (positive control); lane 7 , GST + His-FruE (negative control); lane 8 , GST-FruG + His-FruE; lane 9 , His-FruE (positive control); lane 10 , GST + His-FruK (negative control); lane 11 , GST-FruF + His-FruK; Lane 12 , His-FruK (positive control). IB , immunoblot.

Techniques Used: SDS Page, Staining, Purification, Molecular Weight, Marker, Affinity Column, Incubation, Expressing, Centrifugation, Negative Control, Positive Control

56) Product Images from "Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin β3 Cytoplasmic Tail when Expressed in Mouse Platelets"

Article Title: Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin β3 Cytoplasmic Tail when Expressed in Mouse Platelets

Journal: PLoS ONE

doi: 10.1371/journal.pone.0166136

Interaction of different β3 (WT and mutants) with signaling molecules. (A) Expression of correct truncational mutants in each of the GST-β3 cytoplasmic tail fusion proteins was verified with antibodies specifically recognizing calpain cleaved forms of β3 (Ab 759 and Ab 754) and an antibody recognizing the COOH terminus of (Ab 762). (B) Glutathione-Sepharose 4B beads coated with GST-β3 cytoplasmic tail fusion proteins were incubated overnight with platelet lysates at 4°C. After washing the special antibodies were used to detect talin, kindlin-3, and c-Src binding. Anti-GST antibody was used to verify the loading of the β3 cytoplasmic tail fusion proteins.
Figure Legend Snippet: Interaction of different β3 (WT and mutants) with signaling molecules. (A) Expression of correct truncational mutants in each of the GST-β3 cytoplasmic tail fusion proteins was verified with antibodies specifically recognizing calpain cleaved forms of β3 (Ab 759 and Ab 754) and an antibody recognizing the COOH terminus of (Ab 762). (B) Glutathione-Sepharose 4B beads coated with GST-β3 cytoplasmic tail fusion proteins were incubated overnight with platelet lysates at 4°C. After washing the special antibodies were used to detect talin, kindlin-3, and c-Src binding. Anti-GST antibody was used to verify the loading of the β3 cytoplasmic tail fusion proteins.

Techniques Used: Expressing, Incubation, Binding Assay

57) Product Images from "A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins"

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S58465

Competitive immunoprecipitation assay for Cop1 and Pirh2. Notes: GST-p63 DBD and GST alone were immobilized on glutathione-Sepharose 4B beads and incubated in absence (−) or presence of p28 (+: 10, ++: 100 mole excess), followed by addition of MCF-7 lysates containing Cop1 and Pirh2. Samples were separated by SDS-PAGE and immunoblotted with either anti-Cop1 or anti-Pirh2 antibodies. Lysate: whole-cell lysates of MCF-7 used in assay stably expressed Cop1 and Pirh2. Numbers below Pirh2 bands are the relative percentage to the level of Pirh2 bound to p63 DBD in the absence of p28. Abbreviations: DBD, DNA-binding domain; GST, glutathione S-transferase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IB, immunoblotting.
Figure Legend Snippet: Competitive immunoprecipitation assay for Cop1 and Pirh2. Notes: GST-p63 DBD and GST alone were immobilized on glutathione-Sepharose 4B beads and incubated in absence (−) or presence of p28 (+: 10, ++: 100 mole excess), followed by addition of MCF-7 lysates containing Cop1 and Pirh2. Samples were separated by SDS-PAGE and immunoblotted with either anti-Cop1 or anti-Pirh2 antibodies. Lysate: whole-cell lysates of MCF-7 used in assay stably expressed Cop1 and Pirh2. Numbers below Pirh2 bands are the relative percentage to the level of Pirh2 bound to p63 DBD in the absence of p28. Abbreviations: DBD, DNA-binding domain; GST, glutathione S-transferase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IB, immunoblotting.

Techniques Used: Immunoprecipitation, Incubation, SDS Page, Stable Transfection, Binding Assay, Polyacrylamide Gel Electrophoresis

58) Product Images from "Influenza A Virus NS1 Induces G0/G1 Cell Cycle Arrest by Inhibiting the Expression and Activity of RhoA Protein"

Article Title: Influenza A Virus NS1 Induces G0/G1 Cell Cycle Arrest by Inhibiting the Expression and Activity of RhoA Protein

Journal: Journal of Virology

doi: 10.1128/JVI.03176-12

NS1 can downregulate RhoA activity by direct interaction with RhoA protein. (A) Transfected A549 cell lysates were incubated with equal amounts of GST-Rhotekin bound to glutathione-Sepharose 4B beads. After washing, the bound proteins were analyzed by Western blotting with an anti-RhoA antibody. The data represent the mean fold changes ± the SD of three independent experiments (*, P
Figure Legend Snippet: NS1 can downregulate RhoA activity by direct interaction with RhoA protein. (A) Transfected A549 cell lysates were incubated with equal amounts of GST-Rhotekin bound to glutathione-Sepharose 4B beads. After washing, the bound proteins were analyzed by Western blotting with an anti-RhoA antibody. The data represent the mean fold changes ± the SD of three independent experiments (*, P

Techniques Used: Activity Assay, Transfection, Incubation, Western Blot

59) Product Images from "Functional interaction between the homeoprotein CDX1 and the transcriptional machinery containing the TATA-binding protein"

Article Title: Functional interaction between the homeoprotein CDX1 and the transcriptional machinery containing the TATA-binding protein

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl1034

Characterization of the CDX1/TBP interaction. (A) Interaction of bacterially-produced GST-TBP and cellular HA-CDX1. GST or GST-TBP produced in bacteria was bound to glutathione–Sepharose-4B beads [see also the first two lanes in (B) ] and mixed with HCT116 cells extracts containing HA-CDX1. After GST-pulldown, the presence of GST and GST-TBP was revealed using anti-GST antibody, and the presence of HA-CDX1 was detected using anti-HA antibody. The presence of HA-CDX1 in the cell extracts prior to GST-pulldown was control by western blot with anti-HA antibody (Input). (B) Lack of interaction between bacterially-produced GST-TBP and in vitro translated HA-CDX1. GST (first lane, Coomasie blue staining) and GST-TBP (second lane, Coomasie blue staining) were produced in bacteria and bound to glutathione–Sepharose-4B beads. 35 S-Methionine-labeled HA-CDX1 was synthesized by in vitro transcription/translation (third lane, autoradiography). By mixing GST and HA-CDX1 (fourth lane) or GST-TBP and HA-CDX1 (fifth lane), no interaction was detected after GST-pulldown and autoradiography. (C) CDX1 interaction with components of the TFIID and Med complexes. HCT116 cells were transfected with the control empty vector or with the plasmids encoding HA-TBP or HA-CDX1. Proteins immunoprecipitated with anti-HA antibody were detected by western blots using the antibodies raised against TAF7, TAF12, TAF15 and Med7. D. Effect of ethidium bromide (EtBr) on the CDX1/TBP interaction. HCT116 were transfected with the plasmid encoding HA-CDX1 and the proteins were immunoprecipitated with anti-HA antibody in the presence of increasing amount of ethidium bromide. Western blot with anti-HA antibody was used to control the step of immunoprecipitation and anti-TBP was used to detect the co-immunoprecipitated TBP. The presence of TBP in every cell extract prior to co-immunoprecipitation was controlled using anti-TBP antibody (Input).
Figure Legend Snippet: Characterization of the CDX1/TBP interaction. (A) Interaction of bacterially-produced GST-TBP and cellular HA-CDX1. GST or GST-TBP produced in bacteria was bound to glutathione–Sepharose-4B beads [see also the first two lanes in (B) ] and mixed with HCT116 cells extracts containing HA-CDX1. After GST-pulldown, the presence of GST and GST-TBP was revealed using anti-GST antibody, and the presence of HA-CDX1 was detected using anti-HA antibody. The presence of HA-CDX1 in the cell extracts prior to GST-pulldown was control by western blot with anti-HA antibody (Input). (B) Lack of interaction between bacterially-produced GST-TBP and in vitro translated HA-CDX1. GST (first lane, Coomasie blue staining) and GST-TBP (second lane, Coomasie blue staining) were produced in bacteria and bound to glutathione–Sepharose-4B beads. 35 S-Methionine-labeled HA-CDX1 was synthesized by in vitro transcription/translation (third lane, autoradiography). By mixing GST and HA-CDX1 (fourth lane) or GST-TBP and HA-CDX1 (fifth lane), no interaction was detected after GST-pulldown and autoradiography. (C) CDX1 interaction with components of the TFIID and Med complexes. HCT116 cells were transfected with the control empty vector or with the plasmids encoding HA-TBP or HA-CDX1. Proteins immunoprecipitated with anti-HA antibody were detected by western blots using the antibodies raised against TAF7, TAF12, TAF15 and Med7. D. Effect of ethidium bromide (EtBr) on the CDX1/TBP interaction. HCT116 were transfected with the plasmid encoding HA-CDX1 and the proteins were immunoprecipitated with anti-HA antibody in the presence of increasing amount of ethidium bromide. Western blot with anti-HA antibody was used to control the step of immunoprecipitation and anti-TBP was used to detect the co-immunoprecipitated TBP. The presence of TBP in every cell extract prior to co-immunoprecipitation was controlled using anti-TBP antibody (Input).

Techniques Used: Produced, Western Blot, In Vitro, Staining, Labeling, Synthesized, Autoradiography, Transfection, Plasmid Preparation, Immunoprecipitation

60) Product Images from "NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47"

Article Title: NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl603

Association of NOP132 with DDX18 and DDX47. ( a ) 293 cells were transfected with NOP132 and either FLAG-tagged DDX18 or FLAG-tagged DDX47. Cell lysates were prepared and used for immunoprecipitation with anti-FLAG as described ( 15 ). Total cell protein (2% input) (lanes 1–3). Immunoprecipitates (lanes 4–9). Proteins were detected with anti-NOP132N (upper panels), anti-FLAG (middle panels) or a RAN antibody against the nuclear protein RAN as a loading control (lower panels). Lane 4, control immunoprecipitate (NOP132 transfected); lane 5, control immunoprecipitate (NOP132 transfected) treated with ribonuclease; lane 6, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate; lane 7, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate treated with ribonuclease; lane 8, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate; lane 9, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate treated with ribonuclease. ( b ) 293 cells were transfected with either FLAG-NOP132, FLAG-DDX18, or FLAG-DDX47. Silver-stained 10% SDS–PAGE gel of FLAG-NOP132-, FLAG-DDX47-, or FLAG-DDX18-associated complexes immunoprecipitated with anti-FLAG. Lane 1, molecular weight marker; lane 2, NOP132-associated proteins treated with ribonuclease; lane 3, NOP132-associated proteins; lane 4, DDX47-associated proteins treated with ribonuclease; lane 5, DDX47-associated proteins; lane 6, DDX18-associated proteins treated with ribonuclease; lane 7, DDX18-associated proteins; lane 8, control FLAG tag-associated proteins treated with ribonuclease; lane 9, control FLAG tag-associated proteins. Proteins which were identified by mass spectrometry are shown at the right of the gel image. ( c ) Baculovirus-produced NOP132 was mixed with GST (lane 1), GST-GRWD1 (lane 2), GST-DDX47 (lane 3), or GST-DDX18 (lane 4) bound to the glutathione-Sepharose-4B beads. Bound NOP132 was detected by western blotting using anti-NOP132N (upper panel). GST-fusion proteins stained with Coomassie brilliant blue are shown in the lower panel. Asterisks indicate the positions of the GST-fusion proteins.
Figure Legend Snippet: Association of NOP132 with DDX18 and DDX47. ( a ) 293 cells were transfected with NOP132 and either FLAG-tagged DDX18 or FLAG-tagged DDX47. Cell lysates were prepared and used for immunoprecipitation with anti-FLAG as described ( 15 ). Total cell protein (2% input) (lanes 1–3). Immunoprecipitates (lanes 4–9). Proteins were detected with anti-NOP132N (upper panels), anti-FLAG (middle panels) or a RAN antibody against the nuclear protein RAN as a loading control (lower panels). Lane 4, control immunoprecipitate (NOP132 transfected); lane 5, control immunoprecipitate (NOP132 transfected) treated with ribonuclease; lane 6, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate; lane 7, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate treated with ribonuclease; lane 8, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate; lane 9, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate treated with ribonuclease. ( b ) 293 cells were transfected with either FLAG-NOP132, FLAG-DDX18, or FLAG-DDX47. Silver-stained 10% SDS–PAGE gel of FLAG-NOP132-, FLAG-DDX47-, or FLAG-DDX18-associated complexes immunoprecipitated with anti-FLAG. Lane 1, molecular weight marker; lane 2, NOP132-associated proteins treated with ribonuclease; lane 3, NOP132-associated proteins; lane 4, DDX47-associated proteins treated with ribonuclease; lane 5, DDX47-associated proteins; lane 6, DDX18-associated proteins treated with ribonuclease; lane 7, DDX18-associated proteins; lane 8, control FLAG tag-associated proteins treated with ribonuclease; lane 9, control FLAG tag-associated proteins. Proteins which were identified by mass spectrometry are shown at the right of the gel image. ( c ) Baculovirus-produced NOP132 was mixed with GST (lane 1), GST-GRWD1 (lane 2), GST-DDX47 (lane 3), or GST-DDX18 (lane 4) bound to the glutathione-Sepharose-4B beads. Bound NOP132 was detected by western blotting using anti-NOP132N (upper panel). GST-fusion proteins stained with Coomassie brilliant blue are shown in the lower panel. Asterisks indicate the positions of the GST-fusion proteins.

Techniques Used: Transfection, Immunoprecipitation, Staining, SDS Page, Molecular Weight, Marker, FLAG-tag, Mass Spectrometry, Produced, Western Blot

61) Product Images from "A Cytoplasmic Protein Ssl3829 Is Important for NDH-1 Hydrophilic Arm Assembly in Synechocystis sp. Strain PCC 6803 1"

Article Title: A Cytoplasmic Protein Ssl3829 Is Important for NDH-1 Hydrophilic Arm Assembly in Synechocystis sp. Strain PCC 6803 1

Journal: Plant Physiology

doi: 10.1104/pp.15.01796

Interaction of Ssl3829 with Slr1097. A, GST pull-down assay shows the interaction of Ssl3829 with Slr1097. The expressed proteins were mixed and incubated with Glutathione Sepharose 4B beads on a rotating shaker at 4°C overnight. After washing,
Figure Legend Snippet: Interaction of Ssl3829 with Slr1097. A, GST pull-down assay shows the interaction of Ssl3829 with Slr1097. The expressed proteins were mixed and incubated with Glutathione Sepharose 4B beads on a rotating shaker at 4°C overnight. After washing,

Techniques Used: Pull Down Assay, Incubation

62) Product Images from "MCD1 Associates with FtsZ Filaments via the Membrane-Tethering Protein ARC6 to Guide Chloroplast Division"

Article Title: MCD1 Associates with FtsZ Filaments via the Membrane-Tethering Protein ARC6 to Guide Chloroplast Division

Journal: The Plant Cell

doi: 10.1105/tpc.18.00189

In Vitro Pull-Down Analysis of the Stromal Regions of MCD1 and ARC6. (A) Recombinant His-ARC6 N binds to GST-MCD1 C or GST-MCD1 C(∆277-314) . Glutathione-Sepharose 4B beads were treated with buffer only (lane 2) or coated with GST (lane 3), GST-tagged MCD1 C (lane 4), MCD1C (∆277-314) (lane 5), or MCD1 N (lane 6). The beads were then incubated with crude extracts of E. coli cells expressing His-ARC6 N . Proteins were eluted and analyzed by immunoblotting with anti-His and anti-GST antibodies. (B) Recombinant MBP-PARC6 N -His or His-ARC6 C was not precipitated from crude E. coli extracts by Glutathione-Sepharose 4B beads coated with GST-MCD1 C . All assays were performed more than three times.
Figure Legend Snippet: In Vitro Pull-Down Analysis of the Stromal Regions of MCD1 and ARC6. (A) Recombinant His-ARC6 N binds to GST-MCD1 C or GST-MCD1 C(∆277-314) . Glutathione-Sepharose 4B beads were treated with buffer only (lane 2) or coated with GST (lane 3), GST-tagged MCD1 C (lane 4), MCD1C (∆277-314) (lane 5), or MCD1 N (lane 6). The beads were then incubated with crude extracts of E. coli cells expressing His-ARC6 N . Proteins were eluted and analyzed by immunoblotting with anti-His and anti-GST antibodies. (B) Recombinant MBP-PARC6 N -His or His-ARC6 C was not precipitated from crude E. coli extracts by Glutathione-Sepharose 4B beads coated with GST-MCD1 C . All assays were performed more than three times.

Techniques Used: In Vitro, Recombinant, Incubation, Expressing

63) Product Images from "Conserved Orb6 Phosphorylation Sites Are Essential for Polarized Cell Growth in Schizosaccharomyces pombe"

Article Title: Conserved Orb6 Phosphorylation Sites Are Essential for Polarized Cell Growth in Schizosaccharomyces pombe

Journal: PLoS ONE

doi: 10.1371/journal.pone.0037221

Nak1 and Orb6 interact in vitro and in vivo . A. Extracts from WT (SP199) cells expressing HA-Orb6 and control vector (lane 1), Myc-Nak1 and control vector (lane 2), or HA-Orb6 and Myc-Nak1 (lane 3) from nmt1 promoter expression plasmids were analyzed by western-blots using anti-Myc (9E10) or anti-HA (12CA5) monoclonal antibodies (Input panels). HA-Orb6 was immunoprecipitated from cell extracts with anti-HA antibody and equal portions of the immunoprecipitates were probed with anti-HA antibody and anti-Myc antibody (αHA IP panels). B. Interaction of purified recombinant GST-Orb6 and His 6 -HA-Nak1 was assayed by an in vitro binding assay ( Materials and Methods ). The left panel shows Coomassie blue staining of purified recombinant GST (lane 1), GST-Orb6 (lane 2), His 6 -vector control (lane 3), His 6 -HA-Nak1 (lane 4). Purified His 6 -HA-Nak1 (input, lane 7) was incubated with either GST (lane 5) or GST-Orb6 (lane 6) bound to Glutathione Sepharose 4B beads. The right panel shows a Western blot using anti-HA (12CA5) antibody of His 6 -HA-Nak1 bound to the beads. C. Schematic diagram of mutant Nak1 expression constructs. The Nak1 N-terminal kinase domain (residues 1–262) and C-terminal (CTR) region (554–652) are indicated. The numbers at the left and the bars at the right indicate the region of Nak1 encoded by the various deletion constructs. D. Extracts from wild-type (SP199) cells co-expressing Myc-Orb6 with the control vector (lane 1), HA-Nak1 1–562 (lane 2), HA-Nak1 1–585 (lane 3), HA-Nak1 1–607 (lane 4), HA-Nak1 1–629 (lane 5), HA-Nak1 (lane6), or HA-Nak1 554–652 (lane 7) were analyzed by western-blots using anti-Myc (9E10) or anti-HA (12CA5) monoclonal antibodies (Input panels). Extracts were immunoprecipitated with anti-HA antibody and equal portions of the immunoprecipitates were probed with anti-HA antibody and anti-Myc antibody (αHA IP panels).
Figure Legend Snippet: Nak1 and Orb6 interact in vitro and in vivo . A. Extracts from WT (SP199) cells expressing HA-Orb6 and control vector (lane 1), Myc-Nak1 and control vector (lane 2), or HA-Orb6 and Myc-Nak1 (lane 3) from nmt1 promoter expression plasmids were analyzed by western-blots using anti-Myc (9E10) or anti-HA (12CA5) monoclonal antibodies (Input panels). HA-Orb6 was immunoprecipitated from cell extracts with anti-HA antibody and equal portions of the immunoprecipitates were probed with anti-HA antibody and anti-Myc antibody (αHA IP panels). B. Interaction of purified recombinant GST-Orb6 and His 6 -HA-Nak1 was assayed by an in vitro binding assay ( Materials and Methods ). The left panel shows Coomassie blue staining of purified recombinant GST (lane 1), GST-Orb6 (lane 2), His 6 -vector control (lane 3), His 6 -HA-Nak1 (lane 4). Purified His 6 -HA-Nak1 (input, lane 7) was incubated with either GST (lane 5) or GST-Orb6 (lane 6) bound to Glutathione Sepharose 4B beads. The right panel shows a Western blot using anti-HA (12CA5) antibody of His 6 -HA-Nak1 bound to the beads. C. Schematic diagram of mutant Nak1 expression constructs. The Nak1 N-terminal kinase domain (residues 1–262) and C-terminal (CTR) region (554–652) are indicated. The numbers at the left and the bars at the right indicate the region of Nak1 encoded by the various deletion constructs. D. Extracts from wild-type (SP199) cells co-expressing Myc-Orb6 with the control vector (lane 1), HA-Nak1 1–562 (lane 2), HA-Nak1 1–585 (lane 3), HA-Nak1 1–607 (lane 4), HA-Nak1 1–629 (lane 5), HA-Nak1 (lane6), or HA-Nak1 554–652 (lane 7) were analyzed by western-blots using anti-Myc (9E10) or anti-HA (12CA5) monoclonal antibodies (Input panels). Extracts were immunoprecipitated with anti-HA antibody and equal portions of the immunoprecipitates were probed with anti-HA antibody and anti-Myc antibody (αHA IP panels).

Techniques Used: In Vitro, In Vivo, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Purification, Recombinant, Binding Assay, Staining, Incubation, Mutagenesis, Construct

64) Product Images from "Fructose Uptake in Bifidobacterium longum NCC2705 Is Mediated by an ATP-binding Cassette Transporter *"

Article Title: Fructose Uptake in Bifidobacterium longum NCC2705 Is Mediated by an ATP-binding Cassette Transporter *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.266213

SDS-PAGE and GST pulldown assays to analyze protein-protein interactions of the FruEKFG ABC transporter subunits. A , Coomassie-stained SDS-PAGE of crude extracts ( lanes 1 , 3 , 5 , 7 , and 9 ; 30 μg of protein were loaded per sample) and purified proteins ( lanes 2 , 4 , 6 , 8 , and 10 ; 5–10 μg of purified protein was loaded per sample) of IPTG-induced E. coli BL21(DE3) containing pET32a-FruE ( lanes 1 and 2 ), pET32a-FruK ( lanes 3 and 4 ), pGEX-4T-1-FruF ( lanes 5 and 6 ), pGEX-4T-1-FruG ( lanes 7 and 8 ), and pGEX-4T-1-FruE ( lanes 9 and 10 ). Lane M , molecular weight marker. The proteins were purified by Ni + affinity column (His-FruE and His-FruK) or GST beads (GST-FruF, GST-FruG, and GST-FruE). B , GST pulldown assays probing interactions between FruE or FruK with the membrane permeases FruF and FruG. For pulldown, 25 μg of GST fusion protein was incubated with 5 μl of glutathione-Sepharose 4B beads for 2 h in PBS at 4 °C. Then 200 μl of lysate containing a total of 25 μg of protein of an E. coli BL21 strain expressing the respective His 6 -tagged protein were added, and the bound proteins were precipitated by centrifugation. Lane 1 , GST + His-FruE (negative control); lane 2 , GST-FruF + His-FruE; lane 3 , His-FruE (positive control); lane 4 , GST + His-FruK (negative control); lane 5 , GST-FruF + His-FruK; lane 6 , His-FruK (positive control); lane 7 , GST + His-FruE (negative control); lane 8 , GST-FruG + His-FruE; lane 9 , His-FruE (positive control); lane 10 , GST + His-FruK (negative control); lane 11 , GST-FruF + His-FruK; Lane 12 , His-FruK (positive control). IB , immunoblot.
Figure Legend Snippet: SDS-PAGE and GST pulldown assays to analyze protein-protein interactions of the FruEKFG ABC transporter subunits. A , Coomassie-stained SDS-PAGE of crude extracts ( lanes 1 , 3 , 5 , 7 , and 9 ; 30 μg of protein were loaded per sample) and purified proteins ( lanes 2 , 4 , 6 , 8 , and 10 ; 5–10 μg of purified protein was loaded per sample) of IPTG-induced E. coli BL21(DE3) containing pET32a-FruE ( lanes 1 and 2 ), pET32a-FruK ( lanes 3 and 4 ), pGEX-4T-1-FruF ( lanes 5 and 6 ), pGEX-4T-1-FruG ( lanes 7 and 8 ), and pGEX-4T-1-FruE ( lanes 9 and 10 ). Lane M , molecular weight marker. The proteins were purified by Ni + affinity column (His-FruE and His-FruK) or GST beads (GST-FruF, GST-FruG, and GST-FruE). B , GST pulldown assays probing interactions between FruE or FruK with the membrane permeases FruF and FruG. For pulldown, 25 μg of GST fusion protein was incubated with 5 μl of glutathione-Sepharose 4B beads for 2 h in PBS at 4 °C. Then 200 μl of lysate containing a total of 25 μg of protein of an E. coli BL21 strain expressing the respective His 6 -tagged protein were added, and the bound proteins were precipitated by centrifugation. Lane 1 , GST + His-FruE (negative control); lane 2 , GST-FruF + His-FruE; lane 3 , His-FruE (positive control); lane 4 , GST + His-FruK (negative control); lane 5 , GST-FruF + His-FruK; lane 6 , His-FruK (positive control); lane 7 , GST + His-FruE (negative control); lane 8 , GST-FruG + His-FruE; lane 9 , His-FruE (positive control); lane 10 , GST + His-FruK (negative control); lane 11 , GST-FruF + His-FruK; Lane 12 , His-FruK (positive control). IB , immunoblot.

Techniques Used: SDS Page, Staining, Purification, Molecular Weight, Marker, Affinity Column, Incubation, Expressing, Centrifugation, Negative Control, Positive Control

65) Product Images from "The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation *"

Article Title: The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.721795

The amino acid 43–63 region of SCP1 is responsible for interacting with the N terminus of Twist1. A , GST-tagged full-length SCP1 and nine SCP1 fragments were purified from bacteria using glutathione-Sepharose 4B beads. B , Twist1-F protein was
Figure Legend Snippet: The amino acid 43–63 region of SCP1 is responsible for interacting with the N terminus of Twist1. A , GST-tagged full-length SCP1 and nine SCP1 fragments were purified from bacteria using glutathione-Sepharose 4B beads. B , Twist1-F protein was

Techniques Used: Purification

66) Product Images from "Merkel Cell Polyomavirus Small T Antigen Targets the NEMO Adaptor Protein To Disrupt Inflammatory Signaling"

Article Title: Merkel Cell Polyomavirus Small T Antigen Targets the NEMO Adaptor Protein To Disrupt Inflammatory Signaling

Journal: Journal of Virology

doi: 10.1128/JVI.02159-13

MCPyV ST interacts with NEMO. (A) 293 cells were cotransfected with either EGFP or EGFP-ST and the indicated GST-tagged eukaryotic expression vectors. After 24 h, cell lysates were incubated with GST-Sepharose 4B beads, and interacting proteins were immunoblotted
Figure Legend Snippet: MCPyV ST interacts with NEMO. (A) 293 cells were cotransfected with either EGFP or EGFP-ST and the indicated GST-tagged eukaryotic expression vectors. After 24 h, cell lysates were incubated with GST-Sepharose 4B beads, and interacting proteins were immunoblotted

Techniques Used: Expressing, Incubation

67) Product Images from "A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance ProteinsParasite's Double Play Helps Evade Mouse Immune Response"

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance ProteinsParasite's Double Play Helps Evade Mouse Immune Response

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001358

IRG proteins bind T . gondii virulent strain-derived ROP5 in vitro. Glutathione Sepharose 4B beads loaded at 50 µg protein/100 µl 1∶1 bead suspension with bacterially expressed GST-IRG fusion proteins as bait were incubated at 4°C o/n with whole postnuclear lysates from RH-YFP strain T. gondii . Beads loaded with GST served as negative control. Bound proteins were separated by SDS-PAGE and monoclonal antibody 3E2 was used for detection of ROP5 in subsequent Western blot analysis. (A) In vitro pull-down of ROP5 with bacterially expressed and purified GST-Irga6. A single lysate, equivalent to 50×10 6 organisms per track, was used for GST-Irga6 and GST alone control beads (B) pull-down of ROP5 by Irga6 was markedly enhanced in the presence of 1 mM GDP (left hand blot). Lysates with and without GDP, equivalent to 25×10 6 organisms per track, were prepared from a single batch of T. gondii ; the right hand blot shows equal ROP5 signals from the supernatants of the pull-downs with GST alone with or without nucleotide. T. gondii calnexin provided the loading controls. (C) GST-Irgb6 and GST-Irgb10 also pulled down ROP5, though more weakly than GST-Irga6. One lysate, equivalent to 50×10 6 organisms per track, was used. All four tracks were run on a single gel; the vertical line indicates excision of irrelevant tracks.
Figure Legend Snippet: IRG proteins bind T . gondii virulent strain-derived ROP5 in vitro. Glutathione Sepharose 4B beads loaded at 50 µg protein/100 µl 1∶1 bead suspension with bacterially expressed GST-IRG fusion proteins as bait were incubated at 4°C o/n with whole postnuclear lysates from RH-YFP strain T. gondii . Beads loaded with GST served as negative control. Bound proteins were separated by SDS-PAGE and monoclonal antibody 3E2 was used for detection of ROP5 in subsequent Western blot analysis. (A) In vitro pull-down of ROP5 with bacterially expressed and purified GST-Irga6. A single lysate, equivalent to 50×10 6 organisms per track, was used for GST-Irga6 and GST alone control beads (B) pull-down of ROP5 by Irga6 was markedly enhanced in the presence of 1 mM GDP (left hand blot). Lysates with and without GDP, equivalent to 25×10 6 organisms per track, were prepared from a single batch of T. gondii ; the right hand blot shows equal ROP5 signals from the supernatants of the pull-downs with GST alone with or without nucleotide. T. gondii calnexin provided the loading controls. (C) GST-Irgb6 and GST-Irgb10 also pulled down ROP5, though more weakly than GST-Irga6. One lysate, equivalent to 50×10 6 organisms per track, was used. All four tracks were run on a single gel; the vertical line indicates excision of irrelevant tracks.

Techniques Used: Derivative Assay, In Vitro, Incubation, Negative Control, SDS Page, Western Blot, Purification

68) Product Images from "Swiprosin-1 Is a Novel Actin Bundling Protein That Regulates Cell Spreading and Migration"

Article Title: Swiprosin-1 Is a Novel Actin Bundling Protein That Regulates Cell Spreading and Migration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0071626

Swiprosin-1 dimerizes through its coiled-coil domain, and calcium ions are required for the dimer conformation. (A) HEK293T cells (2×10 6 ) were transiently transfected with 4 µg of GFP, GFP_Swip-1, and myc or increasing concentrations (4, 7, and 11 µg) of myc_Swip-1. The cell lysates were immunoprecipitated with anti-GFP-conjugated beads. Immune complexes were resolved on by SDS-PAGE and blotted with anti-GFP or anti-myc antibodies. (B) HEK293T cells (2×10 6 ) were transiently transfected with GFP, GFP_Swip-1, or mutant GFP_SW1s (M1, M2, and M3). The cell lysates were incubated on ice with glutaraldehyde (GA) at the indicated concentrations (0.001–0.01%) for 20 min. The samples were resolved on SDS-PAGE and blotted with anti-GFP antibodies. The positions of monomer (M), dimer (D), tetramer (T), and GFP alone (G) are indicated by arrows. (C) The purified wild-type His_Swip-1 or wild-type GST_Swip-1 (a) and coiled-coil domain deletion mutants (l, m, and n) were co-incubated with glutathione (GSH)-Sepharose 4B beads for 2 h at 4°C, and the samples were then resolved by SDS-PAGE and blotted with anti-His antibodies (left). Each sample was compared to a loading control (right). (D) The cells from (A) were incubated for 1 h with 20 µM BAPT-AM or 2 µM ionomycin. The cell lysates were immunoprecipitated in the presence of 2 mM EGTA (BAPTA-treated cells) or 1 mM CaCl2 (ionomycin-treated cells), and the amount of binding protein as well as the expression of the indicated proteins were then evaluated by western blotting. All the procedures were performed in the presence of 10 µM cytochalasin D to exclude the effect of actin polymerization. (E) The purified wild-type His_Swip-1 and wild-type GST_Swip-1 (a) or coiled-coil domain containing mutants (h, i) were co-incubated with glutathione (GSH)-Sepharose 4B beads for 2 h at 4°C in the presence or absence of 2 mM EGTA, and the samples were then resolved by SDS-PAGE and blotted with anti-His antibodies (left). Each sample was compared to a loading control (right).
Figure Legend Snippet: Swiprosin-1 dimerizes through its coiled-coil domain, and calcium ions are required for the dimer conformation. (A) HEK293T cells (2×10 6 ) were transiently transfected with 4 µg of GFP, GFP_Swip-1, and myc or increasing concentrations (4, 7, and 11 µg) of myc_Swip-1. The cell lysates were immunoprecipitated with anti-GFP-conjugated beads. Immune complexes were resolved on by SDS-PAGE and blotted with anti-GFP or anti-myc antibodies. (B) HEK293T cells (2×10 6 ) were transiently transfected with GFP, GFP_Swip-1, or mutant GFP_SW1s (M1, M2, and M3). The cell lysates were incubated on ice with glutaraldehyde (GA) at the indicated concentrations (0.001–0.01%) for 20 min. The samples were resolved on SDS-PAGE and blotted with anti-GFP antibodies. The positions of monomer (M), dimer (D), tetramer (T), and GFP alone (G) are indicated by arrows. (C) The purified wild-type His_Swip-1 or wild-type GST_Swip-1 (a) and coiled-coil domain deletion mutants (l, m, and n) were co-incubated with glutathione (GSH)-Sepharose 4B beads for 2 h at 4°C, and the samples were then resolved by SDS-PAGE and blotted with anti-His antibodies (left). Each sample was compared to a loading control (right). (D) The cells from (A) were incubated for 1 h with 20 µM BAPT-AM or 2 µM ionomycin. The cell lysates were immunoprecipitated in the presence of 2 mM EGTA (BAPTA-treated cells) or 1 mM CaCl2 (ionomycin-treated cells), and the amount of binding protein as well as the expression of the indicated proteins were then evaluated by western blotting. All the procedures were performed in the presence of 10 µM cytochalasin D to exclude the effect of actin polymerization. (E) The purified wild-type His_Swip-1 and wild-type GST_Swip-1 (a) or coiled-coil domain containing mutants (h, i) were co-incubated with glutathione (GSH)-Sepharose 4B beads for 2 h at 4°C in the presence or absence of 2 mM EGTA, and the samples were then resolved by SDS-PAGE and blotted with anti-His antibodies (left). Each sample was compared to a loading control (right).

Techniques Used: Transfection, Immunoprecipitation, SDS Page, Mutagenesis, Incubation, Purification, Binding Assay, Expressing, Western Blot

69) Product Images from "Cloning and Characterization of a 2-Cys Peroxiredoxin from Babesiagibsoni"

Article Title: Cloning and Characterization of a 2-Cys Peroxiredoxin from Babesiagibsoni

Journal: The Journal of Veterinary Medical Science

doi: 10.1292/jvms.13-0274

Amino acid sequence alignment and SDS-PAGE analysis of BgTPx-1 protein. (A) Multiple sequence alignment of B. gibsoni BgTPx-1 protein (deduced sequence) with the sequences of other 2-Cys Prxs of apicomplexan parasites. Sequences are from B. bovis (BbTPx-1; XP_001610019), T. gondii (TgPrx; AAG25678), C. parvum (CpTPx; ACV31867) and P. falciparum (BAA97121). Black boxes with white letters show identical residues, and gray boxes with black letters show chemically similar residues. The dashes indicate gaps introduced between the sequences. Two conserved cysteine residues that correspond to Cys47 and Cys170 of the yeast Prx [ 5 ] are marked with asterisks. (B) Expression of BgTPx-1 protein by using the E. coli. expression system and SDS-PAGE analysis. A recombinant plasmid containing the sequence of BgTPx-1 in pGEX-6P1 was transformed in E.coli strain BL21 (DE3), and the transformed colony was cultured in 1 l of LB broth with ampicillin sodium (100 µ g/m l ) at 37°C. When the optical density at 600 n m reached 0.6, expression of the recombinant fusion protein was induced by adding 1 mM isopropyl thio-β-D-galactoside (IPTG) and incubating for another 5 hr at 24°C. The bacterial cultures were lysed with PBS containing 100 µ g/m l lysozyme and 1.5% Triton X-100 with sonication. The supernatant was subjected to protein purification using Glutathione-Sepharose 4B beads and PreScission protease. An SDS-PAGE image of rBgTPx-1 protein is shown. M, protein marker.
Figure Legend Snippet: Amino acid sequence alignment and SDS-PAGE analysis of BgTPx-1 protein. (A) Multiple sequence alignment of B. gibsoni BgTPx-1 protein (deduced sequence) with the sequences of other 2-Cys Prxs of apicomplexan parasites. Sequences are from B. bovis (BbTPx-1; XP_001610019), T. gondii (TgPrx; AAG25678), C. parvum (CpTPx; ACV31867) and P. falciparum (BAA97121). Black boxes with white letters show identical residues, and gray boxes with black letters show chemically similar residues. The dashes indicate gaps introduced between the sequences. Two conserved cysteine residues that correspond to Cys47 and Cys170 of the yeast Prx [ 5 ] are marked with asterisks. (B) Expression of BgTPx-1 protein by using the E. coli. expression system and SDS-PAGE analysis. A recombinant plasmid containing the sequence of BgTPx-1 in pGEX-6P1 was transformed in E.coli strain BL21 (DE3), and the transformed colony was cultured in 1 l of LB broth with ampicillin sodium (100 µ g/m l ) at 37°C. When the optical density at 600 n m reached 0.6, expression of the recombinant fusion protein was induced by adding 1 mM isopropyl thio-β-D-galactoside (IPTG) and incubating for another 5 hr at 24°C. The bacterial cultures were lysed with PBS containing 100 µ g/m l lysozyme and 1.5% Triton X-100 with sonication. The supernatant was subjected to protein purification using Glutathione-Sepharose 4B beads and PreScission protease. An SDS-PAGE image of rBgTPx-1 protein is shown. M, protein marker.

Techniques Used: Sequencing, SDS Page, Expressing, Recombinant, Plasmid Preparation, Transformation Assay, Cell Culture, Sonication, Protein Purification, Marker

70) Product Images from "An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex *"

Article Title: An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.751867

APLF interacts with the Ku·DNA-PKcs·DNA complex. A, His-APLF was immobilized on nitrilotriacetic acid beads and incubated with HeLa whole cell extracts. Beads were washed either in the absence (−) or presence (+) of ethidium bromide (EtBr, 50 μg/ml), then boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to His (for His-APLF), DNA-PKcs, and Ku80 as indicated. B, GST ( lane 2 ) or GST-APLF ( lanes 3–6 ) were immobilized on glutathione-Sepharose 4B beads and incubated with whole cell extracts from HeLa cells that had been either unirradiated (−) or irradiated (10 gray IR) and allowed to recover for 1 h. Beads were washed either in the absence (−) or presence (+) of EtBr (50 μg/ml), then boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs, and Ku80 as indicated. The lower panel represents a longer exposure of the Ku80 blot to show a signal in the input lanes. Lane 1 contained 50 μg of extract from unirradiated cells as a positive control. C, HeLa cells were transiently transfected with FLAG-tagged APLF ( lanes 3 and 4 ) or empty vector ( lane 2 ), then extracts were immunoprecipitated with anti-FLAG antibody, run on SDS-PAGE, and immunoblotted with antibodies to FLAG (for FLAG-APLF), DNA-PKcs and Ku as indicated. Where indicated, ethidium bromide (50 μg/ml) was added to immunoprecipitation wash buffers. Note: a duplicated sample lane has been removed between lanes 2 and 3 . All blots were from the same exposure of the same gels. D, purified DNA-PKcs and/or Ku were incubated with GST-APLF immobilized on glutathione-Sepharose 4B beads in either the absence (−) or presence (+) of CT-DNA (10 μg/ml). Samples were run on SDS-PAGE and immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs and Ku as indicated. E, purified DNA-PKcs and Ku were incubated with GST-APLF ( lanes 3–8 ) or GST ( lane 2 ) immobilized on glutathione-Sepharose 4B beads in the presence of different lengths of DNA (10 μg/ml) and then immunoblotted with antibodies as indicated. In lanes 2 and 4 , proteins were incubated in the presence of 10 μg/ml of CT-DNA, lane 5 contained 40 base ssDNA; lane 6 , 40-bp dsDNA; lane 7 , 100 base ssDNA; and lane 8 , 100-bp dsDNA. Lane 1 contained 100 ng each DNA-PKcs and Ku. Lane 3 contained no DNA. F, purified DNA-PKcs and Ku were incubated with either GST alone ( lanes 2 and 3 ), GST-APLF ( lanes 4 and 5 ), or GST-APLF residues 1–120 ( lanes 6 and 7 ), 110–360 ( lanes 8 and 9 ), or 360–511 ( lanes 9 and 10 ) that had been bound to glutathione-Sepharose 4B beads either in the absence (−) or presence (+) of CT-DNA (80 μg/ml). Samples were washed, run on SDS-PAGE, and immunoblotted. Lane 1 contains 100 ng each DNA-PKcs and Ku. The upper panel is a Ponceau Red-stained membrane, whereas the lower panels show immunoblots for DNA-PKcs and Ku80, respectively. Positions of molecular mass markers (in kDa) are shown on the left-hand side on the Ponceau-stained blot. G, GST alone, GST-APLF, or GST-APLF with mutations of R182E/K183E/R184E or W189G were bound to glutathione-Sepharose 4B beads and incubated with purified DNA-PKcs and Ku in the absence (−) or presence (+) of CT-DNA as above then immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs, and Ku80 as indicated.
Figure Legend Snippet: APLF interacts with the Ku·DNA-PKcs·DNA complex. A, His-APLF was immobilized on nitrilotriacetic acid beads and incubated with HeLa whole cell extracts. Beads were washed either in the absence (−) or presence (+) of ethidium bromide (EtBr, 50 μg/ml), then boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to His (for His-APLF), DNA-PKcs, and Ku80 as indicated. B, GST ( lane 2 ) or GST-APLF ( lanes 3–6 ) were immobilized on glutathione-Sepharose 4B beads and incubated with whole cell extracts from HeLa cells that had been either unirradiated (−) or irradiated (10 gray IR) and allowed to recover for 1 h. Beads were washed either in the absence (−) or presence (+) of EtBr (50 μg/ml), then boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs, and Ku80 as indicated. The lower panel represents a longer exposure of the Ku80 blot to show a signal in the input lanes. Lane 1 contained 50 μg of extract from unirradiated cells as a positive control. C, HeLa cells were transiently transfected with FLAG-tagged APLF ( lanes 3 and 4 ) or empty vector ( lane 2 ), then extracts were immunoprecipitated with anti-FLAG antibody, run on SDS-PAGE, and immunoblotted with antibodies to FLAG (for FLAG-APLF), DNA-PKcs and Ku as indicated. Where indicated, ethidium bromide (50 μg/ml) was added to immunoprecipitation wash buffers. Note: a duplicated sample lane has been removed between lanes 2 and 3 . All blots were from the same exposure of the same gels. D, purified DNA-PKcs and/or Ku were incubated with GST-APLF immobilized on glutathione-Sepharose 4B beads in either the absence (−) or presence (+) of CT-DNA (10 μg/ml). Samples were run on SDS-PAGE and immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs and Ku as indicated. E, purified DNA-PKcs and Ku were incubated with GST-APLF ( lanes 3–8 ) or GST ( lane 2 ) immobilized on glutathione-Sepharose 4B beads in the presence of different lengths of DNA (10 μg/ml) and then immunoblotted with antibodies as indicated. In lanes 2 and 4 , proteins were incubated in the presence of 10 μg/ml of CT-DNA, lane 5 contained 40 base ssDNA; lane 6 , 40-bp dsDNA; lane 7 , 100 base ssDNA; and lane 8 , 100-bp dsDNA. Lane 1 contained 100 ng each DNA-PKcs and Ku. Lane 3 contained no DNA. F, purified DNA-PKcs and Ku were incubated with either GST alone ( lanes 2 and 3 ), GST-APLF ( lanes 4 and 5 ), or GST-APLF residues 1–120 ( lanes 6 and 7 ), 110–360 ( lanes 8 and 9 ), or 360–511 ( lanes 9 and 10 ) that had been bound to glutathione-Sepharose 4B beads either in the absence (−) or presence (+) of CT-DNA (80 μg/ml). Samples were washed, run on SDS-PAGE, and immunoblotted. Lane 1 contains 100 ng each DNA-PKcs and Ku. The upper panel is a Ponceau Red-stained membrane, whereas the lower panels show immunoblots for DNA-PKcs and Ku80, respectively. Positions of molecular mass markers (in kDa) are shown on the left-hand side on the Ponceau-stained blot. G, GST alone, GST-APLF, or GST-APLF with mutations of R182E/K183E/R184E or W189G were bound to glutathione-Sepharose 4B beads and incubated with purified DNA-PKcs and Ku in the absence (−) or presence (+) of CT-DNA as above then immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs, and Ku80 as indicated.

Techniques Used: Incubation, SDS Page, Irradiation, Positive Control, Transfection, Plasmid Preparation, Immunoprecipitation, Purification, Staining, Western Blot

71) Product Images from "Viral Interferon Regulatory Factor 1 of Kaposi's Sarcoma-Associated Herpesvirus Interacts with a Translocation Liposarcoma Protein-Associated Serine-Arginine Protein"

Article Title: Viral Interferon Regulatory Factor 1 of Kaposi's Sarcoma-Associated Herpesvirus Interacts with a Translocation Liposarcoma Protein-Associated Serine-Arginine Protein

Journal: Osong Public Health and Research Perspectives

doi: 10.1016/j.phrp.2012.01.001

Interactions of vIRF1 with TASR1 or 2 in vivo. (A) 293 T cells were cotransfected with GST or GST-vIRF1 and Flag- TASR1 expression plasmids. Cells were lysed with EBC buffer, and cell extracts incubated with glutathione sepharose 4B beads. GST fusion and Flag fusion proteins were detected using western blotting with anti-GST (top panel) and anti-Flag antibodies (bottom panel), respectively. Lanes 1, 3, GST and Flag-TASR1; 3, 4, GST-vIRF1 and Flag-TASR1 (B) 293 T cells were cotransfected with GST or GST-vIRF1 and Flag-TASR2 expression plasmids and subjected to similar experiments as those specified in Panel A. Lanes 5, 7, GST and Flag-TASR2; Lanes 6, 8, GST-vIRF1 and Flag-TASR2. (C) Reciprocal assay for Panel A. 293 T cells were cotransfected with GST or GST-vIRF1 and Flag-TASR1 expression plasmids. Cell extracts were incubated with anti-Flag antibody and precipitated with protein G-Sepharose beads for 1 h. GST fusion and Flag fusion proteins were detected by western blotting with anti-GST and anti-Flag antibodies respectively. Lanes 1, 3, GST and Flag-TASR1; Lanes 2, 4, GST-vIRF1 and Flag-TASR1 (D) Reciprocal assay for Panel B. 293 T cells were cotransfected with GST or GST-vIRF1 expression plasmid and Flag-TASR2, and subjected to similar experiments as those specified in panel C. Lanes 5, 7, GST and Flag-TASR1; Lanes 6, 8, GST-vIRF1 and Flag-TASR1.
Figure Legend Snippet: Interactions of vIRF1 with TASR1 or 2 in vivo. (A) 293 T cells were cotransfected with GST or GST-vIRF1 and Flag- TASR1 expression plasmids. Cells were lysed with EBC buffer, and cell extracts incubated with glutathione sepharose 4B beads. GST fusion and Flag fusion proteins were detected using western blotting with anti-GST (top panel) and anti-Flag antibodies (bottom panel), respectively. Lanes 1, 3, GST and Flag-TASR1; 3, 4, GST-vIRF1 and Flag-TASR1 (B) 293 T cells were cotransfected with GST or GST-vIRF1 and Flag-TASR2 expression plasmids and subjected to similar experiments as those specified in Panel A. Lanes 5, 7, GST and Flag-TASR2; Lanes 6, 8, GST-vIRF1 and Flag-TASR2. (C) Reciprocal assay for Panel A. 293 T cells were cotransfected with GST or GST-vIRF1 and Flag-TASR1 expression plasmids. Cell extracts were incubated with anti-Flag antibody and precipitated with protein G-Sepharose beads for 1 h. GST fusion and Flag fusion proteins were detected by western blotting with anti-GST and anti-Flag antibodies respectively. Lanes 1, 3, GST and Flag-TASR1; Lanes 2, 4, GST-vIRF1 and Flag-TASR1 (D) Reciprocal assay for Panel B. 293 T cells were cotransfected with GST or GST-vIRF1 expression plasmid and Flag-TASR2, and subjected to similar experiments as those specified in panel C. Lanes 5, 7, GST and Flag-TASR1; Lanes 6, 8, GST-vIRF1 and Flag-TASR1.

Techniques Used: In Vivo, Expressing, Incubation, Western Blot, Plasmid Preparation

72) Product Images from "RABL2 interacts with the intraflagellar transport-B complex and CEP19 and participates in ciliary assembly"

Article Title: RABL2 interacts with the intraflagellar transport-B complex and CEP19 and participates in ciliary assembly

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E17-01-0017

Interaction between RABL2 and CEP19. (A) Interaction of RABL2 with CEP19. HEK293T cells were transiently cotransfected with expression vectors for EGFP-CEP19 and RABL2B(WT)-HA or its mutant (S35N, Q80L, or D73G). At 24 h after the transfection, lysates were prepared from the transfected cells and immunoprecipitated with GST-fused anti–GFP Nb prebound to glutathione–Sepharose 4B beads. Proteins bound to the precipitated beads were subjected to SDS–PAGE and immunoblotting analysis using anti-HA or anti-GFP antibodies. (B) Schematic representation of the structures of CEP19 and its deletion constructs. (C) RABL2 interacts with the C-terminal region of CEP19. Lysates prepared from HEK293T cells transfected with expression vectors for RABL2B-HA and EGFP, or EGFP-tagged CEP19(WT) or its deletion construct, as indicated, were processed for immunoprecipitation with GST–anti-GFP Nb, followed by immunoblotting analysis, as described for A.
Figure Legend Snippet: Interaction between RABL2 and CEP19. (A) Interaction of RABL2 with CEP19. HEK293T cells were transiently cotransfected with expression vectors for EGFP-CEP19 and RABL2B(WT)-HA or its mutant (S35N, Q80L, or D73G). At 24 h after the transfection, lysates were prepared from the transfected cells and immunoprecipitated with GST-fused anti–GFP Nb prebound to glutathione–Sepharose 4B beads. Proteins bound to the precipitated beads were subjected to SDS–PAGE and immunoblotting analysis using anti-HA or anti-GFP antibodies. (B) Schematic representation of the structures of CEP19 and its deletion constructs. (C) RABL2 interacts with the C-terminal region of CEP19. Lysates prepared from HEK293T cells transfected with expression vectors for RABL2B-HA and EGFP, or EGFP-tagged CEP19(WT) or its deletion construct, as indicated, were processed for immunoprecipitation with GST–anti-GFP Nb, followed by immunoblotting analysis, as described for A.

Techniques Used: Expressing, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Construct

73) Product Images from "SNAP23/25 and VAMP2 mediate exocytic event of transferrin receptor-containing recycling vesicles"

Article Title: SNAP23/25 and VAMP2 mediate exocytic event of transferrin receptor-containing recycling vesicles

Journal: Biology Open

doi: 10.1242/bio.012146

Interaction of exocyst subunits with SNAP23 and SNAP25. (A) HEK293T cells were cotransfected with expression vectors for either EGFP-SNAP23 (lanes 1-8) or EGFP-SNAP25 (lanes 9-16) and C-terminally tRFP-tagged exocyst subunit indicated, except for Sec6, which was N-terminally tRFP-tagged (lanes 3 and 11). Lysates prepared from the cotransfected cells were subjected to immunoprecipitation (IP) using GST-tagged anti-GFP Nanobody prebound to glutathione–Sepharose 4B beads, followed by immunoblot (IB) analysis using anti-tRFP (upper panels) or anti-GFP (lower panels) antibody. (B) HEK293T cells were cotransfected with expression vectors for either EGFP-SNAP23 (lanes 1-5) or EGFP-SNAP25 (lanes 6-10) and an exocyst subunit indicated (lanes 1-5 and lanes 6-10) with either an N-terminal (lanes 1, 4, 5, 9 and 10) or C-terminal (lanes 2, 3 and 6-8) mCherry-tag. Lysates prepared from the cotransfected cells were subjected to immunoprecipitation (IP) using GST–anti-GFP Nanobody prebound to glutathione-Sepharose 4B beads and following immunoblot (IB) analysis using anti-RFP (upper panel) or anti-GFP (lower panel) antibody. (C) HEK293T cells were transfected with an expression vector for EGFP-SNAP23 or -SNAP25, and lysates prepared from the transfected cells were subjected to immunoprecipitation using GST–anti-GFP Nanobody prebound to glutathione–Sepharose 4B beads, followed by immunoblot analysis using antibody against Sec8 (lanes 1-6) or Exo70 (lanes 7-12).
Figure Legend Snippet: Interaction of exocyst subunits with SNAP23 and SNAP25. (A) HEK293T cells were cotransfected with expression vectors for either EGFP-SNAP23 (lanes 1-8) or EGFP-SNAP25 (lanes 9-16) and C-terminally tRFP-tagged exocyst subunit indicated, except for Sec6, which was N-terminally tRFP-tagged (lanes 3 and 11). Lysates prepared from the cotransfected cells were subjected to immunoprecipitation (IP) using GST-tagged anti-GFP Nanobody prebound to glutathione–Sepharose 4B beads, followed by immunoblot (IB) analysis using anti-tRFP (upper panels) or anti-GFP (lower panels) antibody. (B) HEK293T cells were cotransfected with expression vectors for either EGFP-SNAP23 (lanes 1-5) or EGFP-SNAP25 (lanes 6-10) and an exocyst subunit indicated (lanes 1-5 and lanes 6-10) with either an N-terminal (lanes 1, 4, 5, 9 and 10) or C-terminal (lanes 2, 3 and 6-8) mCherry-tag. Lysates prepared from the cotransfected cells were subjected to immunoprecipitation (IP) using GST–anti-GFP Nanobody prebound to glutathione-Sepharose 4B beads and following immunoblot (IB) analysis using anti-RFP (upper panel) or anti-GFP (lower panel) antibody. (C) HEK293T cells were transfected with an expression vector for EGFP-SNAP23 or -SNAP25, and lysates prepared from the transfected cells were subjected to immunoprecipitation using GST–anti-GFP Nanobody prebound to glutathione–Sepharose 4B beads, followed by immunoblot analysis using antibody against Sec8 (lanes 1-6) or Exo70 (lanes 7-12).

Techniques Used: Expressing, Immunoprecipitation, Transfection, Plasmid Preparation

74) Product Images from "p28, A first in class peptide inhibitor of cop1 binding to p53"

Article Title: p28, A first in class peptide inhibitor of cop1 binding to p53

Journal: British Journal of Cancer

doi: 10.1038/bjc.2013.266

p28-binding regions on p53. ( A ) Purified various fragments of p53 and GST alone were used for a GST pull-down assay. Each sample was analysed by immunoblotting with an anti-p28 antibody. ( B ) Competitive pull-down assay. Immobilised GST-p53 1–393 and GST alone on glutathione-Sepharose 4B beads were incubated in absence (−) or presence of p28 (+: 10, ++: 100 mole excess), followed by adding MCF-7 lysates containing COP1. Samples were separated by SDS-PAGE and immunoblotted with an anti-COP1 antibody. Lysate: whole-cell lysates of MCF-7 used for the assay showed the stable expression of COP1. Numbers below COP1 bands were relative percentage to the level of COP1 bound to p53 in absence of p28. ( C ) COP1 binding to various fragments of p53 in absence or presence of p28 was analysed using a similar competitive pull-down assay. Immobilised various fragments of p53 and GST alone were treated with or without p28 (100 mole excess), followed by incubation with MCF-7 lysates containing COP1.
Figure Legend Snippet: p28-binding regions on p53. ( A ) Purified various fragments of p53 and GST alone were used for a GST pull-down assay. Each sample was analysed by immunoblotting with an anti-p28 antibody. ( B ) Competitive pull-down assay. Immobilised GST-p53 1–393 and GST alone on glutathione-Sepharose 4B beads were incubated in absence (−) or presence of p28 (+: 10, ++: 100 mole excess), followed by adding MCF-7 lysates containing COP1. Samples were separated by SDS-PAGE and immunoblotted with an anti-COP1 antibody. Lysate: whole-cell lysates of MCF-7 used for the assay showed the stable expression of COP1. Numbers below COP1 bands were relative percentage to the level of COP1 bound to p53 in absence of p28. ( C ) COP1 binding to various fragments of p53 in absence or presence of p28 was analysed using a similar competitive pull-down assay. Immobilised various fragments of p53 and GST alone were treated with or without p28 (100 mole excess), followed by incubation with MCF-7 lysates containing COP1.

Techniques Used: Binding Assay, Purification, Pull Down Assay, Incubation, SDS Page, Expressing

75) Product Images from "Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ"

Article Title: Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddy322

PC1-CTT binds to TAZ without disrupting the TAZ-14-3-3 interaction. (A) HEK293 cells were co-transfected with FLAG-TAZ or FLAG-YAP and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies. (B) HEK293 cells were co-transfected with FLAG-TAZ and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-HA sepharose, then blotted with the indicated antibodies. (C) A GST-tagged construct containing the C-terminal 91 amino acids of the PC1-CTT (p91) was produced in BL21 bacteria and purified on glutathione-sepharose 4B beads. The GST-p91 coated glutathione beads were then exposed to lysates from HEK293 cells expressing FLAG-TAZ and the resulting complexes were blotted with the indicated antibodies. (D) HEK293 cells were co-transfected with FLAG-TAZ(WT) or FLAG-TAZ(S89A) and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies.
Figure Legend Snippet: PC1-CTT binds to TAZ without disrupting the TAZ-14-3-3 interaction. (A) HEK293 cells were co-transfected with FLAG-TAZ or FLAG-YAP and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies. (B) HEK293 cells were co-transfected with FLAG-TAZ and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-HA sepharose, then blotted with the indicated antibodies. (C) A GST-tagged construct containing the C-terminal 91 amino acids of the PC1-CTT (p91) was produced in BL21 bacteria and purified on glutathione-sepharose 4B beads. The GST-p91 coated glutathione beads were then exposed to lysates from HEK293 cells expressing FLAG-TAZ and the resulting complexes were blotted with the indicated antibodies. (D) HEK293 cells were co-transfected with FLAG-TAZ(WT) or FLAG-TAZ(S89A) and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies.

Techniques Used: Transfection, Immunoprecipitation, Construct, Produced, Purification, Expressing

Related Articles

Transduction:

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Article Snippet: The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins. .. The lysate and bead eluates were subjected to Western blotting against the following antibodies: 0.2 μg/ml anti-GST (Santa Cruz B-14), 0.1 μg/ml anti-Flag M2 (Sigma), 0.1 μg/ml anti-HA (Sigma) and 2.0 μg/ml mouse anti-protein phosphatase 5 (BD Transduction Laboratories).

Clone Assay:

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Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Approximately 20 independent clones were screened to test NHE51D4 expression by Western blot and immunofluorescence microscopy, and several independent clones expressing moderate levels of NHE5 were analyzed. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: GST-RARα2 was obtained from E. coli cells transformed with an appropriate RARα2 cDNA construct cloned in the EcoRI-Not1 sites of pGEX4T2 . .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Centrifugation:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Beads were then washed 3 times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (Invitrogen).

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
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Article Snippet: .. This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech). .. After a 30-min incubation (4°C, rotation), the beads were washed four times with 20 vol of washing buffer (20 mM Hepes, pH 7.4, 0.1% CHAPS, 0.15 mM NaCl).

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: The lysates were cleared by centrifugation, and the supernatants, containing the His-tagged proteins, were incubated with Ni-NTA agarose (Qiagen, Hilden, Germany) for 1 h at 4°C. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

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Article Title:
Article Snippet: Cells expressing GST-tagged nuclear transport factors, GST-tagged NLSs, and GST-AID were harvested by centrifugation and lysed by sonication in buffer A, B, or C respectively ( supplemental Table S2 ). .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Luciferase:

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor
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Filtration:

Article Title:
Article Snippet: Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Peak fractions were further purified by anion exchange at pH 8.0 on a Q column (GE Healthcare) and subsequent gel filtration on Superdex 75 (GE Healthcare).

Stable Transfection:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: NHE51D4 was transfected to PC12 cells using the conventional calcium phosphate method , and cells stably expressing NHE51D4 were selected in selection media containing G418 (200 μg/ml). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Synthesized:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator. .. After overnight incubation, the beads were washed three times, 10 min each with 0.1 M PBS.

Construct:

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
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Electrophoresis:

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Incubation:

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Article Snippet: .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C. .. GST Pulldown —A 35 S-labeled NHE5 C-terminal domain (Gly491 -Leu896 ) was produced by in vitro transcription-translation using the T n T-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: The pellet was suspended in extraction buffer (20 mM Hepes, pH 7.4, 1% CHAPS, 0.15 M NaCl) and incubated on ice for 20 min. .. This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech).

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: The lysates were cleared by centrifugation, and the supernatants, containing the His-tagged proteins, were incubated with Ni-NTA agarose (Qiagen, Hilden, Germany) for 1 h at 4°C. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours. .. Pulled-down proteins were subjected to WB analysis using anti-HA, anti-RARα or anti-GST antibodies.

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. .. For the kinase kinase assay, the AMPKK preparation was incubated with 10 μl of a 50% slurry of the glutathione-Sepharose beads with bound GST-AMPKα1, plus 200 μM AMP, 200 μM ATP, 5 mM MgCl2 in assay buffer in a final volume of 25 μl.

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins. .. The beads were washed extensively with lysis buffer followed by phosphate-buffered saline buffer.

Article Title:
Article Snippet: .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. E. coli -expressing recombinant His-CTNNBL1 and His-CTNNBL1(Δ1–76) were lysed by sonication in buffer D (Table S2 ), and the recombinant proteins were purified by binding onto a nickel-nitrilotriacetic acid column (GE Healthcare) followed by elution with a 0–500 mm imidazole gradient.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation. ..

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. For the in vitro pVHL ubiquitination assay, reaction mixtures were incubated 0.5 μg of His-E1, 0.2 μg of UCP, 2 μg of pVHL and 1.25 μg Flag-ubiquitin in reaction buffer along with the ATP regeneration system at 37°C for 1 h. To precipitate the ubiquitinated pVHL, the total reaction mixtures were pulled down with Glutathione Sepharose beads or Ni-NTA agarose on a rotary shaker at 4°C for 3 h. The beads were then washed three times under reducing (1% NP40 in NET gel buffer) or non-reducing (4 M urea and 1% NP40 in NET gel buffer) conditions and resuspended in 2X SDS sample buffer under denaturing conditions. .. For the in vivo ubiquitination assay, cells were co-transfected with indicated plasmids and treated with 10 μM MG132 for 12 h before harvesting.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator. .. After overnight incubation, the beads were washed three times, 10 min each with 0.1 M PBS.

Expressing:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Co-affinity Purification Experiments and Western Blot Analysis To perform co-affinity purification experiments, cloned ORFs were transferred from pDONR207 to pDEST27 expression vector (Invitrogen) to achieve GST fusion, and to pCI-neo-3xFLAG vector for 3xFLAG fusion. .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Protein expression was induced by incubating transformed BL21 Escherichia coli cells with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside at 37 °C for 3 h. E. coli cells were collected by centrifugation and resuspended in lysis buffer containing 1% Triton X-100 and protease inhibitor mixture (Roche Diagnostics, Laval, Canada) in PBS. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: 5 ml of an overnight culture grown in selective medium containing 1% glucose was used to inoculate 100 ml fresh selective medium containing 2% galactose to induce the expression of the GST fusion proteins. .. This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech).

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Paragraph title: Recombinant Protein Expression and Protein Purification ... Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers.

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *
Article Snippet: Paragraph title: Protein Expression in Escherichia coli ... GST-ICP27 , GST-Nup62, GST-RaeI, and GST alone recombinant proteins were expressed in E. coli BL21 cells and purified on glutathione-Sepharose beads (GE Healthcare) as described ( ).

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: .. The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. ..

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor
Article Snippet: .. Materials GST expression vector pGEX-4T-1 and Glutathione-Sepharose 4B beads were purchased from Amersham Biosciences (Piscataway, NJ). .. The E. coli BL21 gold strain was purchased from Stratagene (La Jolla, CA).

Article Title:
Article Snippet: Cells expressing GST-tagged nuclear transport factors, GST-tagged NLSs, and GST-AID were harvested by centrifugation and lysed by sonication in buffer A, B, or C respectively ( supplemental Table S2 ). .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Bradford Assay:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers. .. The concentrations of the proteins were measured by Bradford assay (Bio Rad) using Ultrospec® 3000 spectrophotometer (Pharmacia Biotech).

Western Blot:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Paragraph title: Co-affinity Purification Experiments and Western Blot Analysis ... Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Approximately 20 independent clones were screened to test NHE51D4 expression by Western blot and immunofluorescence microscopy, and several independent clones expressing moderate levels of NHE5 were analyzed. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours. .. Pulled-down proteins were subjected to WB analysis using anti-HA, anti-RARα or anti-GST antibodies.

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: A portion of the cell lysate was saved for Western blotting to indicate protein signal input. .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Transformation Assay:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Protein expression was induced by incubating transformed BL21 Escherichia coli cells with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside at 37 °C for 3 h. E. coli cells were collected by centrifugation and resuspended in lysis buffer containing 1% Triton X-100 and protease inhibitor mixture (Roche Diagnostics, Laval, Canada) in PBS. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: GST-RARα2 was obtained from E. coli cells transformed with an appropriate RARα2 cDNA construct cloned in the EcoRI-Not1 sites of pGEX4T2 . .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Kinase Assay:

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: .. The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. ..

Derivative Assay:

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: For the pull-down experiments [ ], we used the described GST-RARα1 , GST-RARα2 and derived recombinant proteins. .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Transfection:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Two days after transfection, HEK-293T cells were washed in PBS, then resuspended in lysis buffer (0.5% Nonidet P-40, 20 mM Tris–HCl at pH 8, 120 mM NaCl and 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail (Roche). .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: NHE51D4 was transfected to PC12 cells using the conventional calcium phosphate method , and cells stably expressing NHE51D4 were selected in selection media containing G418 (200 μg/ml). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours. .. Pulled-down proteins were subjected to WB analysis using anti-HA, anti-RARα or anti-GST antibodies.

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: Forty-eight hours after transfection, cells were harvested in ice-cold phosphate-buffered saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EGTA, and supplemented with complete protease inhibitors [Roche], 1 mM Na3 VO4 , 1 μM microcystin LR, and 5 mM β-glycerophosphate). .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Concentration Assay:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: The purified proteins were then dissolved in SDS sample buffer and separated by SDS-PAGE to analyze their concentration and purity. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: .. The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. ..

Protease Inhibitor:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Two days after transfection, HEK-293T cells were washed in PBS, then resuspended in lysis buffer (0.5% Nonidet P-40, 20 mM Tris–HCl at pH 8, 120 mM NaCl and 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail (Roche). .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Protein expression was induced by incubating transformed BL21 Escherichia coli cells with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside at 37 °C for 3 h. E. coli cells were collected by centrifugation and resuspended in lysis buffer containing 1% Triton X-100 and protease inhibitor mixture (Roche Diagnostics, Laval, Canada) in PBS. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Polymerase Chain Reaction:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Expression and Purification of GST Fusion Proteins —For producing GST fusion proteins, PCR fragments corresponding to different regions of the SCAMP2 cytoplasmic domains were inserted into a pGEX-2T bacterial expression vector (Amersham Biosciences) in-frame with the N-terminal GST tag as described previously ( ). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Sonication:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Cell lysates were then incubated for 30 min on ice and then sonicated four times for 30 s. After sonication, cell debris was cleared by centrifugation for 10 min at 16,000 × g at 4 °C. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: To purify GST-tagged recombinant proteins, cells were lysed in lysis buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 and 1 mM PMSF, pH 7.4) by sonication. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title:
Article Snippet: Cells expressing GST-tagged nuclear transport factors, GST-tagged NLSs, and GST-AID were harvested by centrifugation and lysed by sonication in buffer A, B, or C respectively ( supplemental Table S2 ). .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation. ..

Affinity Purification:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences). .. Cell culture and transient transfection HEK-293T and HeLa cells were cultured at 37°C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, NY, USA) and 1X (v/v) antibiotics (Gibco).

Binding Assay:

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: Paragraph title: GST pull-down binding assay. ... The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Article Title:
Article Snippet: Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. E. coli -expressing recombinant His-CTNNBL1 and His-CTNNBL1(Δ1–76) were lysed by sonication in buffer D (Table S2 ), and the recombinant proteins were purified by binding onto a nickel-nitrilotriacetic acid column (GE Healthcare) followed by elution with a 0–500 mm imidazole gradient.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation. .. To control for nonspecific binding in the GST pulldown experiment, in vitro-translated product was first gently agitated with glutathione-Sepharose beads in NETN buffer (20 mM Tris, pH 8, 1 mM EDTA, 100 mM NaCl, 0.5% NP-40, 2 μg of aprotinin per ml, 1 μg of pepstatin A per ml, 1 mM phenylmethylsulfonyl fluoride, and 2 μg of leupeptin per ml) for 30 min at 4°C, followed by gentle agitation with GST-coupled glutathione-Sepharose beads for 1 h at 4°C.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Paragraph title: In vitro Binding Assay ... Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator.

Immunofluorescence:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Approximately 20 independent clones were screened to test NHE51D4 expression by Western blot and immunofluorescence microscopy, and several independent clones expressing moderate levels of NHE5 were analyzed. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Nucleic Acid Electrophoresis:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Purified complexes and protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% NuPAGE Bis–Tris gels with MOPS running buffer (Invitrogen), and transferred to a nitrocellulose membrane.

Microscopy:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Approximately 20 independent clones were screened to test NHE51D4 expression by Western blot and immunofluorescence microscopy, and several independent clones expressing moderate levels of NHE5 were analyzed. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Purification:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Paragraph title: Co-affinity Purification Experiments and Western Blot Analysis ... Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C. .. GST Pulldown —A 35 S-labeled NHE5 C-terminal domain (Gly491 -Leu896 ) was produced by in vitro transcription-translation using the T n T-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: Paragraph title: Purification of GST-tagged proteins from the yeast, electrophoresis, and immunoblot analysis ... This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech).

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences). .. Cell culture and transient transfection HEK-293T and HeLa cells were cultured at 37°C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, NY, USA) and 1X (v/v) antibiotics (Gibco).

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers. .. The purified proteins were dialyzed using Slide-A-Lyzer dialysis cassette (Thermo Scientific).

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *
Article Snippet: .. GST-ICP27 , GST-Nup62, GST-RaeI, and GST alone recombinant proteins were expressed in E. coli BL21 cells and purified on glutathione-Sepharose beads (GE Healthcare) as described ( ). .. Histidine-tagged ICP27 from pET-ICP27 was expressed in BL21 DE3 and purified using Talon resin (Clontech) as described ( ).

Article Title:
Article Snippet: Recombinant Protein Purification Recombinant proteins were purified from Escherichia coli BL21(DE3) transformants that had been incubated at 16 °C in LB overnight following induction with 1 mm isopropyl 1-thio-β-d -galactopyranoside at an A 600 of 0.6. .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation. ..

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator. .. After overnight incubation, the beads were washed three times, 10 min each with 0.1 M PBS.

Article Title: The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions
Article Snippet: .. Glutathione S -transferase (GST) pull-down assays GST fusion proteins purified from Escherichia coli LE392 or E.coli BL21-Star(DE3)pLysS (Invitrogen) extracts using glutathione–sepharose beads (Amersham Pharmacia Biotech) were used in pull-down assays as described previously ( ). .. Transient transfection assays NIH 3T3 fibroblasts (passage 123) (ATCC CRL 1658) were cultured in DMEM supplemented with 10% calf serum (HyClone, Logan, UT), penicillin (100 U/ml) and 100 μg/ml streptomycin (Life Technologies, Inc.).

Protein Purification:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Paragraph title: Recombinant Protein Expression and Protein Purification ... Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers.

Article Title:
Article Snippet: Paragraph title: Recombinant Protein Purification ... Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Protein Extraction:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: Paragraph title: Recombinant protein extraction ... The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Positron Emission Tomography:

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *
Article Snippet: GST-ICP27 , GST-Nup62, GST-RaeI, and GST alone recombinant proteins were expressed in E. coli BL21 cells and purified on glutathione-Sepharose beads (GE Healthcare) as described ( ). .. Histidine-tagged ICP27 from pET-ICP27 was expressed in BL21 DE3 and purified using Talon resin (Clontech) as described ( ).

SDS Page:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Purified complexes and protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% NuPAGE Bis–Tris gels with MOPS running buffer (Invitrogen), and transferred to a nitrocellulose membrane.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech). .. Samples for immunoblotting were separated by SDS-PAGE (12% acrylamide, except where noted), electrotransferred to nitrocellulose, and probed with the appropriate primary antibodies according to standard protocols ( ).

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: The purified proteins were then dissolved in SDS sample buffer and separated by SDS-PAGE to analyze their concentration and purity. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Plasmid Preparation:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Briefly, 5×105 HEK-293T cells were dispensed in each well of a 6-well plate, and transfected 24 h later with 500 ng of each pDEST27 plasmid encoding viral ORFs and 300 ng of pCI-neo-3xFLAG vector containing 3xFLAG-tagged indicated proteins. .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Expression and Purification of GST Fusion Proteins —For producing GST fusion proteins, PCR fragments corresponding to different regions of the SCAMP2 cytoplasmic domains were inserted into a pGEX-2T bacterial expression vector (Amersham Biosciences) in-frame with the N-terminal GST tag as described previously ( ). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: Far-western and GST pull-down assays COS-7 cells were transfected with a pcDNA3 plasmid containing haemoagglutinin (HA)-tagged RARa 2 (pHA-RARα2 ). .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor
Article Snippet: .. Materials GST expression vector pGEX-4T-1 and Glutathione-Sepharose 4B beads were purchased from Amersham Biosciences (Piscataway, NJ). .. The E. coli BL21 gold strain was purchased from Stratagene (La Jolla, CA).

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: HVS ORF73 constructs were in vitro transcribed and translated with 5 μg of plasmid template and either 35 S-labeled methionine or 14 C-labeled leucine (PerkinElmer). .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: In vitro Binding Assay Plasmid pHA.DX3 DNA (0.8 μg) was used to synthesize radio-labeled DDX3 protein in vitro by utilizing a TNT T7 Coupled Reticulocyte Lysate System (Promega) in the presence of 30 μCi of [35 S]-methionine (Perkin Elmer). .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator.

Ubiquitin Assay:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. For the in vitro pVHL ubiquitination assay, reaction mixtures were incubated 0.5 μg of His-E1, 0.2 μg of UCP, 2 μg of pVHL and 1.25 μg Flag-ubiquitin in reaction buffer along with the ATP regeneration system at 37°C for 1 h. To precipitate the ubiquitinated pVHL, the total reaction mixtures were pulled down with Glutathione Sepharose beads or Ni-NTA agarose on a rotary shaker at 4°C for 3 h. The beads were then washed three times under reducing (1% NP40 in NET gel buffer) or non-reducing (4 M urea and 1% NP40 in NET gel buffer) conditions and resuspended in 2X SDS sample buffer under denaturing conditions. .. For the in vivo ubiquitination assay, cells were co-transfected with indicated plasmids and treated with 10 μM MG132 for 12 h before harvesting.

Recombinant:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: Paragraph title: Recombinant protein extraction ... The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Paragraph title: Recombinant Protein Expression and Protein Purification ... Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers.

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *
Article Snippet: .. GST-ICP27 , GST-Nup62, GST-RaeI, and GST alone recombinant proteins were expressed in E. coli BL21 cells and purified on glutathione-Sepharose beads (GE Healthcare) as described ( ). .. Histidine-tagged ICP27 from pET-ICP27 was expressed in BL21 DE3 and purified using Talon resin (Clontech) as described ( ).

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours. .. Pulled-down proteins were subjected to WB analysis using anti-HA, anti-RARα or anti-GST antibodies.

Article Title:
Article Snippet: Paragraph title: Recombinant Protein Purification ... Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

In Vitro:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C. .. GST Pulldown —A 35 S-labeled NHE5 C-terminal domain (Gly491 -Leu896 ) was produced by in vitro transcription-translation using the T n T-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: Paragraph title: In vitro translation and GST pulldown assays. ... For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation.

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. For the in vitro pVHL ubiquitination assay, reaction mixtures were incubated 0.5 μg of His-E1, 0.2 μg of UCP, 2 μg of pVHL and 1.25 μg Flag-ubiquitin in reaction buffer along with the ATP regeneration system at 37°C for 1 h. To precipitate the ubiquitinated pVHL, the total reaction mixtures were pulled down with Glutathione Sepharose beads or Ni-NTA agarose on a rotary shaker at 4°C for 3 h. The beads were then washed three times under reducing (1% NP40 in NET gel buffer) or non-reducing (4 M urea and 1% NP40 in NET gel buffer) conditions and resuspended in 2X SDS sample buffer under denaturing conditions. .. For the in vivo ubiquitination assay, cells were co-transfected with indicated plasmids and treated with 10 μM MG132 for 12 h before harvesting.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator. .. After overnight incubation, the beads were washed three times, 10 min each with 0.1 M PBS.

Selection:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: NHE51D4 was transfected to PC12 cells using the conventional calcium phosphate method , and cells stably expressing NHE51D4 were selected in selection media containing G418 (200 μg/ml). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Spectrophotometry:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers. .. The concentrations of the proteins were measured by Bradford assay (Bio Rad) using Ultrospec® 3000 spectrophotometer (Pharmacia Biotech).

Produced:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C. .. GST Pulldown —A 35 S-labeled NHE5 C-terminal domain (Gly491 -Leu896 ) was produced by in vitro transcription-translation using the T n T-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions.

Activation Assay:

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: .. The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. ..

Lysis:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Two days after transfection, HEK-293T cells were washed in PBS, then resuspended in lysis buffer (0.5% Nonidet P-40, 20 mM Tris–HCl at pH 8, 120 mM NaCl and 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail (Roche). .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Protein expression was induced by incubating transformed BL21 Escherichia coli cells with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside at 37 °C for 3 h. E. coli cells were collected by centrifugation and resuspended in lysis buffer containing 1% Triton X-100 and protease inhibitor mixture (Roche Diagnostics, Laval, Canada) in PBS. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: To purify GST-tagged recombinant proteins, cells were lysed in lysis buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 and 1 mM PMSF, pH 7.4) by sonication. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: Forty-eight hours after transfection, cells were harvested in ice-cold phosphate-buffered saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EGTA, and supplemented with complete protease inhibitors [Roche], 1 mM Na3 VO4 , 1 μM microcystin LR, and 5 mM β-glycerophosphate). .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Chick Chorioallantoic Membrane Assay:

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor
Article Snippet: Materials GST expression vector pGEX-4T-1 and Glutathione-Sepharose 4B beads were purchased from Amersham Biosciences (Piscataway, NJ). .. CaM and G protein Gβ antibodies were purchased from Upstate Biotechnology (Charlottesville, VA).

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    GE Healthcare glutathione sepharose beads
    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with <t>glutathione-sepharose</t> beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.
    Glutathione Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Recombinant, Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Protein Concentration, Silver Staining, Western Blot, Incubation, Imaging

    GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Blocking Assay, Silver Staining, Quantitation Assay, Western Blot, Imaging, Incubation

    GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Mass Spectrometry, Isolation, Liquid Chromatography with Mass Spectroscopy

    GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Mass Spectrometry, Cell Culture

    GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Expressing, Lysis, Incubation, SDS Page, Staining, Silver Staining, Mutagenesis, Infection, Activity Assay, Reporter Assay

    GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Staining, Binding Assay, Transfection, Expressing, Construct, Positive Control, SDS Page, Silver Staining, Infection, Activity Assay, Reporter Assay, Western Blot, Imaging