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. 2011 Jul 19;286(38):32962–32975. doi: 10.1074/jbc.M111.243899

FIGURE 7.

FIGURE 7.

Palmitoylation of AKAP79 is required for interaction with AC8 and down-regulation of SOCE-stimulated AC8 activity. A, co-immunoprecipitation of AKAP79-YFP with AC8-HA. Mutant (C36S,C129S (C36,129S)) or wild type (Wt) AKAP79-YFP were expressed in cells stably expressing HA-AC8 (+) or wild type HEK-293 cells (−). AC8-HA pulldowns were performed using HA affinity beads, and AKAP79 and AC8-HA were detected by Western blot. Phospho-AC8 was detected using a phospho-PKA substrate-specific antibody. IB, immunoblot. B, AKAP79 inputs used to precipitate AC8-HA were blotted to measure the total expression of the proteins. C, densitometry analysis of pulldown blots in A (AKAP79 relative to AC8-HA). Data represent mean ± S.E. of four experiments. *, p < 0.05. TG, thapsigargin. D, pulldown using HA antibody not covalently coupled to agarose beads. AC8-HA and phospho-AC8 was detected by Western blot. E, inputs used to precipitate AC8-HA were blotted to measure the total expression of AC8 and AKAP79 proteins. F, densitometry analysis of pulldown blots (phospho-AC8-HA relative to AC8-HA) (mean ± S.E. of five separate experiments). n.s., not significant. G, SOCE-mediated increases in cAMP production measured with the FRET-based sensor Epac2-camps in HEK-AC8 cells expressing empty vector (control), wild type AKAP79-HA or AKAP79-YFP, or palmitoylation mutant (AKAP79-YFP C36S,C129S). Data are plotted as relative FRET ratio changes with F0 taken at 240 s, and values normalized to the maximum signal seen following stimulation with a mixture to saturate the cAMP sensor (10 μm forskolin, 10 μm isoproterenol, 10 μm PGE1, and 100 μm 3-isobutyl-1-methylxanthine). Average traces are plotted from 34 to 54 individual cells. H, input samples for AC8-HA pulldowns were also examined for ERK phosphorylation using a pERK-specific antibody. Tubulin was used a loading control. Densitometry data compares the effects of AKAP79 (Wt or double mutant) and SOCE stimulation of AC8 on pERK signals. Data are representative of mean ± S.E. from four similar experiments, **, p < 0.01; ***, p < 0.001.