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

Figure 5.

Figure 5

Anchored PKA is required for the association of 14-3-3 with AKAP-Lbc. (A) HEK-293 cells expressing the Flag-tagged fusions of AKAP-Lbc (lanes 1–3) or of its mutants A1251P/I1260P (lanes 4–6) and S1565A (lanes 7–9) 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). Cell lysates were subjected to immunoprecipitation with anti-Flag antibodies. Western blots of the immunoprecipitates and of the cell extracts were revealed using anti-Flag monoclonal antibodies to detect Flag-AKAP-Lbc (upper panel) or anti-14-3-3β rabbit polyclonal antibodies to detect the endogenous 14-3-3β (middle and lower panels). (B) Densitometry of the bands corresponding to 14-3-3β co-immunoprecipitated with AKAP-Lbc from cells that were treated as indicated in (A). 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.