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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: Biochim Biophys Acta. 2009 Jul 23;1790(10):1179–1190. doi: 10.1016/j.bbagen.2009.07.012

Figure 2. PKCδ interacts with and phosphorylates p190 FF and GTPase domains.

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

Panel a, schematic representation of GST fused p190 FF and p190 GTPase domains. Panel b, bacterially expressed proteins encompassing the GST fused p190 FF (∼60kDa) or p190 GTPase domains (∼54kDa), or GST protein (∼27kDa) were purified, resolved by SDS-PAGE and visualized via Coomassie blue staining. The purified GST-fused proteins were subsequently incubated with equivalent amounts LMVEC lysates (panel c) or equivalent amounts of LMVEC lysates containing increasing NaCl concentrations (panel d). Protein precipitates were resolved via SDS-PAGE and membranes were immunoblotted for PKCδ. In parallel, endothelial cell (EC) lysates were resolved as positive control for immunoblot analysis of PKCδ. Panel e, GST, GST p190 FF or GST p190 GTPase proteins were incubated with (+) or without (−) recombinant, enzymatically active PKCδ. In parallel, enzymatically active PKCδ was incubated in the absence of any exogenous substrate (No substrate). All reactions were incubated for 30min at 30°C. Proteins were resolved on SDS-PAGE and membranes autoradiographed. Solid arrows indicate phosphorylated p190 domains. Dashed arrow indicates PKCδ autophosphorylation. Representative gels or immunoblots are shown in panels b-d and representative autoradiograph is shown in panel e. n = 2−5.