Figure 6
Figure 6. PSTPIP1-R405C has impaired interaction with WASP but not PTP-PEST. (A) Anti-GFP antibody was used to immunoprecipitate GFP or GFP-tagged PSTPIP1 constructs from the indicated THP-1 rescue cell lines. Blots were probed with anti-GFP antibody and anti-WASP antibody to visualize the coimmunoprecipitation (IP) of WASP. Equivalent loading was determined by probing a blot of the lysates as above. (B) His-tagged WASP or GST-tagged PSTPIP1 proteins (labeled as GST-WT, GST-R405C, and GST-ΔSH3) were purified from E coli. They were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue to assess purity. (C) Pulldown assay using the purified proteins described in (B). Soluble His-WASP was incubated with the indicated bead-bound GST-PSTPIP1 construct. Blots were probed for WASP and GST (to detect PSTPIP1) to determine the degree of interaction. The amount of WASP pulled down by the GST-PSTPIP1 mutants was normalized to the interaction with GST-PSTPIP1-WT. (D) Anti-GFP antibody was used to coimmunoprecipitate PTP-PEST with GFP or GFP-tagged PSTPIP1 constructs from the indicated THP-1 rescue cell lines. Anti–PTP-PEST and anti-GFP antibodies were used to probe the immunoprecipitation and input blots. (E) Quantification of the amount of PTP-PEST coimmunoprecipitating with PSTPIP1 was determined as a percent of input and normalized to the PSTPIP1-WT value. (F) Anti-GFP antibody was used to immunoprecipitate GFP-PSTPIP1-WT or -R405C from THP-1 rescue cells. Immunoprecipitates and lysates were probed with anti-phosphotyrosine (4G10 platinum) and anti-GFP antibodies to assess PSTPIP1 phosphorylation. (G) Quantification of the phosphorylation of PSTPIP1-WT and -R405C is shown relative to WT. Unlabeled arrowheads indicate the band(s) of interest in each panel. Labeled arrowheads indicate the locations of the molecular weight markers (kDa). All values are mean ± SEM from 3 (C,G) or 4 (E) independent experiments. **P < .01; ****P < .0001 as determined by one-way ANOVA with Sidak’s multiple comparisons test (C,E) or paired Student t test (G). IB, immunoblotting. NS, nonsignificant.

PSTPIP1-R405C has impaired interaction with WASP but not PTP-PEST. (A) Anti-GFP antibody was used to immunoprecipitate GFP or GFP-tagged PSTPIP1 constructs from the indicated THP-1 rescue cell lines. Blots were probed with anti-GFP antibody and anti-WASP antibody to visualize the coimmunoprecipitation (IP) of WASP. Equivalent loading was determined by probing a blot of the lysates as above. (B) His-tagged WASP or GST-tagged PSTPIP1 proteins (labeled as GST-WT, GST-R405C, and GST-ΔSH3) were purified from E coli. They were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue to assess purity. (C) Pulldown assay using the purified proteins described in (B). Soluble His-WASP was incubated with the indicated bead-bound GST-PSTPIP1 construct. Blots were probed for WASP and GST (to detect PSTPIP1) to determine the degree of interaction. The amount of WASP pulled down by the GST-PSTPIP1 mutants was normalized to the interaction with GST-PSTPIP1-WT. (D) Anti-GFP antibody was used to coimmunoprecipitate PTP-PEST with GFP or GFP-tagged PSTPIP1 constructs from the indicated THP-1 rescue cell lines. Anti–PTP-PEST and anti-GFP antibodies were used to probe the immunoprecipitation and input blots. (E) Quantification of the amount of PTP-PEST coimmunoprecipitating with PSTPIP1 was determined as a percent of input and normalized to the PSTPIP1-WT value. (F) Anti-GFP antibody was used to immunoprecipitate GFP-PSTPIP1-WT or -R405C from THP-1 rescue cells. Immunoprecipitates and lysates were probed with anti-phosphotyrosine (4G10 platinum) and anti-GFP antibodies to assess PSTPIP1 phosphorylation. (G) Quantification of the phosphorylation of PSTPIP1-WT and -R405C is shown relative to WT. Unlabeled arrowheads indicate the band(s) of interest in each panel. Labeled arrowheads indicate the locations of the molecular weight markers (kDa). All values are mean ± SEM from 3 (C,G) or 4 (E) independent experiments. **P < .01; ****P < .0001 as determined by one-way ANOVA with Sidak’s multiple comparisons test (C,E) or paired Student t test (G). IB, immunoblotting. NS, nonsignificant.

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