Effects of α-actinin on αIIbβ3 inside-out signaling in CMK cells. Human megakaryoblastic CMK cells were transiently transfected with plasmids encoding for α-actinin and Tac subunit of the human interleukin-2 receptor. Protein expression and integrin activation were assessed 20 hours after transfection. (A) Intracellular α-actinin and surface αIIbβ3 were determined by flow cytometry. Bar charts represent specific antibody binding to highly transfected cells (CD25-allophycocyanin fluorescence > 50) normalized to mock plasmid (pcDNA3.1)–transfected cells. Transfected CMK cells were incubated with PAR1-AP (50μM) for the time indicated. αIIbβ3 was immunoprecipitated then immunoblotted with anti–α-actinin antibody(B). Bar charts represent PAC-1 binding to wild-type α-actinin (C) or mutant α-actinin (D) transfected cells. Error bars represent SEMs of 3 experiments. *P < .05.