Coengagement of PECAM-1 with FcγRIIa cross-linking with IV.3 results in dephosphorylation of PLCγ2. (A, top panel) Washed platelets were stimulated by FcγRIIa cross-linking with and without coengagement of PECAM-1 over 5 minutes at 37°C with stirring. Immunoprecipitation of PLCγ2 with polyclonal anti-PLCγ2 antibody followed by immunoblotting with HRP-conjugated antiphosphotyrosine antibody (RC20) and ECL development. (A, bottom panel) Blot was stripped and reprobed for PLCγ2 antigen content with PLCγ2 antibody. (B, top panel) Same experimental conditions as in panel A, with the exception that coengagement of CD151 was performed. (B, bottom panel) Blot was stripped and reprobed for PLCγ2 antigen content with PLCγ2 antibody. (C, top panel) Similar experimental conditions as in panel A, with the exception that a single 1-minute timepoint and a dose response with Neutravidin (0-100 μg/mL) were performed. (C, bottom panel) Blot was stripped and reprobed for PLCγ2 antigen content with PLCγ2 antibody.