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. 2011 Mar 3;286(17):15073–15084. doi: 10.1074/jbc.M110.213660

FIGURE 5.

FIGURE 5.

CIN85 is a phosphosubstrate of classical PKCs in response to FcγRIIa cross-linking. A, neutrophils (20 × 106 cells/ml) were incubated with 1 μm calyculin for the indicated times at room temperature. Whole cell lysates were analyzed by SDS-PAGE and immunoblotted for CIN85 and flotillin-1 (loading control). B, neutrophils (20 × 106 cells/ml) were stimulated with 100 nm phorbol 12-myristate 13-acetate (PMA) or DMSO for 45 s at room temperature. CIN85 was immunoprecipitated as described under “Experimental Procedures,” and the immunoprecipitates were probed for a phospho-(Ser)PKC substrate or CIN85. The graph represents the densitometric ratio of the phospho-(Ser)PKC substrate signal over the total CIN85 signal. C, neutrophils (20 × 106 cells/ml) were incubated in the presence or absence of Gö6976 (1 μm) for 10 min before FcγRIIa cross-linking at room temperature for 1 min. CIN85 was immunoprecipitated as described under “Experimental Procedures,” and the immunoprecipitates were probed for phospho-(Ser)PKC substrates or CIN85. The histogram represents the quantification of the mean densitometric readings of the phospho-(Ser)PKC substrate signal (relative to the total CIN85 signal) observed in three independent experiments. WB, Western blot; IP, immunoprecipitation; Error bars, S.E.