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. 2004 Jul 1;23(14):2811–2820. doi: 10.1038/sj.emboj.7600287

Figure 2.

Figure 2

Interaction between 14-3-3 and AKAP-Lbc is regulated by PKA. (A) GST or GST-AKAP-Lbc 1388–1922 was incubated for 1 h in the absence (No PKA) or presence (+PKA) of purified PKA catalytic subunit and electroblotted. The membranes were incubated with 50 nM of S-tagged 14-3-3β (left panel) and HRP-conjugated S-protein and revealed by autoradiography. A protein staining showing the amount of GST and GST-AKAP-Lbc 1388–1922 used in the assay is shown (right panel). (B) HeLa-S3 cells were serum starved for 24 h and then treated for 10 min in the absence or presence of 10 μM forskolin (FSK) or 10 μM forskolin+20 μM of myristoylated PKI 5–24 peptide (FSK+PKImyr). Lysates were subjected to immunoprecipitation with either nonimmune IgG or anti-AKAP-Lbc polyclonal antibodies. Immunoblots were revealed using anti-AKAP-Lbc polyclonal antibodies (upper panel), anti-RIIα monoclonal antibodies (middle panel) and anti-14-3-3β monoclonal antibodies. (C) Densitometry of the bands corresponding to 14-3-3β co-immunoprecipitated with AKAP-Lbc from cells that were treated as indicated in (B). The amount of 14-3-3β in the immunoprecipitates was normalized to the 14-3-3β content of the cell extracts. Results are the mean±s.e. of three independent experiments.