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. Author manuscript; available in PMC: 2010 Jul 21.
Published in final edited form as: Biochemistry. 2009 Jul 21;48(28):6674–6683. doi: 10.1021/bi900322a

Figure 3. PKC phosphorylates α1c Ser1517, Ser1674 and Ser1842/1843.

Figure 3

A, The schematic demonstrates the PKC phosphorylation sites within the GST fusion protein 1509-1905. B, upper panel, Shown is autoradiogram of PKCα in vitro kinase reaction performed with [γ-32P] ATP and GST-fused 1509-1905, 1509-1642, 1642-1778, 1776-1905. Lower panel, Coomassie-staining of autoradiogram demonstrating amount of fusion protein used. C, upper panels, Shown are autoradiograms of PKCα in vitro kinase reactions performed with [γ-32P] ATP and WT and Ala-substituted GST-fused 1509-1642, 1642-1778, 1776-1905 proteins. Arrowheads indicate full-length GST fusion protein; lower bands are truncated GST proteins. Lower panel, Coomassie-staining of autoradiogram demonstrating amount of fusion protein used. D, WT GST-fused 1509-1905 fusion proteins were phosphorylated with PKCα, size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose and immunoblotted using anti-phospho-Ser1517, Ser1674 and Ser1842/1843 antibodies (pS1517, pS1674 and pS1842/43 respectively). PKCα phosphorylates Ser1517, Ser1674 and Ser1842/1843. E, Extracts from HEK cells transfected with WT α1c + β2a subunits were prepared, followed by pre-immune (PI) or α1c immunoprecipitation and PKCα kinase reaction as indicated. Samples were size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with anti-phospho-Ser1517, Ser1674 and Ser1842/1843 antibodies (upper panel) or α1c antibody (lower panel). F, Shown is pS1674 immunoblot of in vitro kinase reactions of eight PKC isoforms. All blots are representative of 3 or more similar experiments.