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. 2010 Jan 6;30(1):242–254. doi: 10.1523/JNEUROSCI.4933-08.2010

Figure 2.

Figure 2.

PKC phosphorylates SNAP-25 in heterologous cells. a, HEK-293 cells overexpressing WT and S187A SNAP-25 were treated with TPA (100 nm, 15 min) in the absence or presence of Bis. Cell lysates were subjected to Western blotting and probed with an antibody that recognizes p-SNAP-25 (top). TPA induced phosphorylation of WT, but not mutant SNAP-25(S187A). Stripping and reprobing with an antibody against total SNAP-25 reveals equal loading of samples and the absence of SNAP-25 in mock-transfected cells (bottom). b–g, HEK-293 cells overexpressing WT SNAP-25 and SNAP-25(S187A) were fixed, permeabilized and colabeled with antibodies to p-SNAP-25 and total SNAP-25, followed by Cy3 (red, p-SNAP-25) and AMCA (blue, SNAP-25)-conjugated secondary antibodies, respectively. b, Under basal conditions, p-SNAP-25 is at background intensity. c, TPA markedly promotes phosphorylation of SNAP-25, particularly evident at the leading edges of the cell. d, Bis (1 μm) abolished TPA-induced phosphorylation of SNAP-25. e, f, TPA does not promote phosphorylation of mutant SNAP-25(S187A). g, Summary bar graph of results shown in b–f. n = 8–12 for each condition; ***p < 0.001.