The capacity of Ptsn to support integrin activation depends on binding to PP1 c . (A) Schematic of β-Ptsn indicating the PP1c- binding domain containing a KISF motif. (B) Mutation of the KISF residues to four Ala (4A) in Ptsn blocks its interaction with PP1c. 293A cells were transfected with plasmids encoding EGFP-tagged PP1c in combination with either β-Ptsn WT or 4A mutant. Ptsn was immunoprecipitated (IP) using an anti-Flag antibody before immunoblotting. (C and D) 293A cells that express constitutively active α_IIb(R995A)β₃ were transduced with a lentivirus encoding a shRNA against Ptsn and transfected with a plasmid encoding EGFP-tagged shRNA-resistant Ptsn either WT or 4A mutant. (C) 293A cells that express constitutively active α_IIb(R995A)β₃ were transduced with a lentivirus encoding a shRNA against Ptsn. A scrambled shRNA was used as a negative control. Cells were then transfected with a plasmid encoding DsRed-tagged shRNA-resistant β-Ptsn either WT or 4A mutant. Integrin activation was measured by flow cytometry using the monoclonal antibody PAC1 that specifically recognizes the activated form of α_IIbβ₃. The activation index was calculated as (F − Fo)/(Fm − Fo), where F is the 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. (D) The expression of 4A Ptsn was confirmed by immunoblotting. Source data are available for this figure: SourceData F7.