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. Author manuscript; available in PMC: 2009 Apr 24.
Published in final edited form as: Cell Commun Adhes. 2007;14(5):211–226. doi: 10.1080/15419060701755958

Figure 3.

Figure 3

Cx43 is a substrate for Akt and for 14-3-3-binding in vivo. 14-3-3 θ was not detected in immunoprecipitates (IP) of Cx43 from EGF-treated Rat-1 cells lysed in a 0.5% NP-40 buffer, as shown by Western blotting (bottom blot on the left-hand side of (a)). Cx43 that remained in the 0.5% NP-40-insoluble fraction was solubilized with hot SDS sample buffer and was recognized on Western blots by an antibody to phospho-Ser “mode-1”14-3-3-binding sites and an antibody to phosphorylated Akt-substrates (right side of (a)), as shown by stripping and reprobing the blot. The protein bands recognized by the motif antibodies migrated in the same position as Cx43. 14-3-3 θ was also detected in the 0.5% NP-40 insoluble fraction (bottom blot on the right hand side of (a)). (b) Akt was activated in the EGF-treated cells, as shown by the phosphorylation of Ser473 of Akt on a protein in the whole cell lysate (WCL) that migrated as Akt-3 and was recognized by an Akt-3 specific antibody.

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