Figure 7
Figure 7. Effect of myr-RGT on the interaction of integrin β3 cytoplasmic domain with Src or talin. (A) Platelets preincubated with 250 μM of myr-RGT or scrambled myr-GRT were lysed with lysis buffer, and the lysates of untreated or peptide-treated platelets were analyzed with an immunoprecipitation procedure as follows. The lysates were incubated with SZ21 antibody or nonspecific mouse IgG. After washing, the immune complexes were subjected to SDS-PAGE and probed by Western blotting using monoclonal antibodies SZ22 or 327 against integrin αIIb or c-Src, respectively (lanes 1-5). In another set of experiments, immunoprecipitation was performed with monoclonal antibody 327 and blotted with SZ21 or 327 (lanes 6-10). Representative results of 3 experiments are shown. (B) Glutathione-Sepharose 4B beads coated with GST-wild-type integrin β3 cytoplasmic tail fusion protein were incubated overnight with purified His-Src-SH3 in the presence of peptides as indicated. After wash, protein complexes were subjected to Western blot analysis with anti-His or anti-GST antibodies. Peptide concentrations: *62.5 μM; **125 μM; ***250 μM; ****500 μM. (C) Increasing concentrations of purified GST-Src-SH3 or GST protein were added to the microtiter wells coated with RGT or GRT peptide (20 μg/mL). Binding of the purified proteins to the peptides was detected by incubation with mouse anti-GST antibody, followed by horseradish peroxidase-conjugated antimouse Ig antibody. Specific binding was normalized by subtracting the OD (optical density) values of the blank wells from that of the sample wells. Results were presented as percentage of the maximal binding. Data were organized as binding of GST-Src-SH3 to RGT peptide (●), GST-Src-SH3 to GRT peptide (■), GST protein to RGT peptide (△), and GST protein to GRT peptide (□). (D) Glutathione-Sepharose 4B beads coated with GST-integrin β3 cytoplasmic tail fusion proteins were incubated overnight with platelet lysates in the presence of peptides as indicated at 4°C before being lysed by SDS sample buffer. Talin was detected with the monoclonal antibody 8d4. Anti-GST antibody binding was used to verify the loading of the β3 cytoplasmic tail fusion proteins. The increased electrophoretic mobility of GST-β3-741 documents the 21 residue truncation of this fusion protein.

Effect of myr-RGT on the interaction of integrin β3 cytoplasmic domain with Src or talin. (A) Platelets preincubated with 250 μM of myr-RGT or scrambled myr-GRT were lysed with lysis buffer, and the lysates of untreated or peptide-treated platelets were analyzed with an immunoprecipitation procedure as follows. The lysates were incubated with SZ21 antibody or nonspecific mouse IgG. After washing, the immune complexes were subjected to SDS-PAGE and probed by Western blotting using monoclonal antibodies SZ22 or 327 against integrin αIIb or c-Src, respectively (lanes 1-5). In another set of experiments, immunoprecipitation was performed with monoclonal antibody 327 and blotted with SZ21 or 327 (lanes 6-10). Representative results of 3 experiments are shown. (B) Glutathione-Sepharose 4B beads coated with GST-wild-type integrin β3 cytoplasmic tail fusion protein were incubated overnight with purified His-Src-SH3 in the presence of peptides as indicated. After wash, protein complexes were subjected to Western blot analysis with anti-His or anti-GST antibodies. Peptide concentrations: *62.5 μM; **125 μM; ***250 μM; ****500 μM. (C) Increasing concentrations of purified GST-Src-SH3 or GST protein were added to the microtiter wells coated with RGT or GRT peptide (20 μg/mL). Binding of the purified proteins to the peptides was detected by incubation with mouse anti-GST antibody, followed by horseradish peroxidase-conjugated antimouse Ig antibody. Specific binding was normalized by subtracting the OD (optical density) values of the blank wells from that of the sample wells. Results were presented as percentage of the maximal binding. Data were organized as binding of GST-Src-SH3 to RGT peptide (●), GST-Src-SH3 to GRT peptide (■), GST protein to RGT peptide (△), and GST protein to GRT peptide (□). (D) Glutathione-Sepharose 4B beads coated with GST-integrin β3 cytoplasmic tail fusion proteins were incubated overnight with platelet lysates in the presence of peptides as indicated at 4°C before being lysed by SDS sample buffer. Talin was detected with the monoclonal antibody 8d4. Anti-GST antibody binding was used to verify the loading of the β3 cytoplasmic tail fusion proteins. The increased electrophoretic mobility of GST-β3-741 documents the 21 residue truncation of this fusion protein.

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