Figure 2.
Ptsn regulates integrin activation. Silencing of Ptsn reduces integrin activation. (A) Domain organization of α-Ptsn (short) and β-Ptsn (long) isoforms. (B) Both isoforms of Ptsn associate with RIAM. 293A cells were transfected with plasmids encoding Flag-tagged α- or β-Ptsn in combination with vectors encoding EGFP-RIAM or EGFP. Immunoblots were used to analyze proteins binding to an anti-Flag affinity matrix. IP, immunoprecipitation. (C) Both isoforms of Ptsn restore integrin activation in Ptsn-silenced cells. 293A cells that express constitutively active αIIb(R995A)β3 were transduced with a lentivirus encoding an shRNA against Ptsn. A scrambled shRNA was used as a negative control. Cells were then transfected with a plasmid encoding either EGFP-tagged shRNA resistant α- or β-Ptsn or EGFP alone. Integrin activation was measured by flow cytometry using the monoclonal antibody PAC1 that specifically recognizes the activated form of αIIbβ3. The activation index was calculated as (F − Fo)/(Fm − Fo), where F is the geometric mean fluorescence intensity (MFI) of PAC1 binding, Fo is the MFI in presence of the competitive inhibitor integrilin, and Fm is the MFI upon addition of the integrin-activating anti-LIBS6 antibody. Bar graphs represent mean ± SEM (n = 3 independent experiments). One-way ANOVA with Tukey post-test; **, P < 0.01; ***, P < 0.001. In C, the expression of Ptsn was confirmed by immunoblotting. (D) Expression of spinophilin does not complement the loss of Ptsn. The expression of Ptsn was silenced in αIIb(R995A)β3-expressing 293A cells, and the activation index was determined as in C in cells transfected with Ds Red β-Ptsn or spinophilin (SPN). Bar graphs represent mean ± SEM (n = 3 independent experiments). One-way ANOVA with Tukey post-test; **, P < 0.01; ***, P < 0.001. (E) Protein expression was assessed by immunoblotting. Source data are available for this figure: SourceData F2.

Ptsn regulates integrin activation. Silencing of Ptsn reduces integrin activation. (A) Domain organization of α-Ptsn (short) and β-Ptsn (long) isoforms. (B) Both isoforms of Ptsn associate with RIAM. 293A cells were transfected with plasmids encoding Flag-tagged α- or β-Ptsn in combination with vectors encoding EGFP-RIAM or EGFP. Immunoblots were used to analyze proteins binding to an anti-Flag affinity matrix. IP, immunoprecipitation. (C) Both isoforms of Ptsn restore integrin activation in Ptsn-silenced cells. 293A cells that express constitutively active αIIb(R995A)β3 were transduced with a lentivirus encoding an shRNA against Ptsn. A scrambled shRNA was used as a negative control. Cells were then transfected with a plasmid encoding either EGFP-tagged shRNA resistant α- or β-Ptsn or EGFP alone. Integrin activation was measured by flow cytometry using the monoclonal antibody PAC1 that specifically recognizes the activated form of αIIbβ3. The activation index was calculated as (F − Fo)/(Fm − Fo), where F is the geometric mean fluorescence intensity (MFI) of PAC1 binding, Fo is the MFI in presence of the competitive inhibitor integrilin, and Fm is the MFI upon addition of the integrin-activating anti-LIBS6 antibody. Bar graphs represent mean ± SEM (n = 3 independent experiments). One-way ANOVA with Tukey post-test; **, P < 0.01; ***, P < 0.001. In C, the expression of Ptsn was confirmed by immunoblotting. (D) Expression of spinophilin does not complement the loss of Ptsn. The expression of Ptsn was silenced in αIIb(R995A)β3-expressing 293A cells, and the activation index was determined as in C in cells transfected with Ds Red β-Ptsn or spinophilin (SPN). Bar graphs represent mean ± SEM (n = 3 independent experiments). One-way ANOVA with Tukey post-test; **, P < 0.01; ***, P < 0.001. (E) Protein expression was assessed by immunoblotting. Source data are available for this figure: SourceData F2.

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