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Fig. 2. Surface expression of K_ATP channels is impaired by 14-3-3 sequestration. (A) Surface expression in oocytes of K_ATP channels assembled from independently expressed Kir6.2HA and SUR1 in the absence (black bars) or presence (grey bars) of the 14-3-3-scavenger pGpLI-R18 (n=50, nine batches of oocytes; P<0.0001). (B) Mean steady-state whole-cell currents recorded from control INS-1 cells (n=6) and INS-1 cells expressing pGpLI-R18 (n=6; P<0.05). ATP concentration-inhibition relationships for K_ATP currents were almost identical for the two groups of cells (data not shown). (C) Coimmunoprecipitation of 14-3-3 proteins and K_ATP channels. Cells were treated with the crosslinker DSS where indicated. (Top and middle panels) Cell extracts (1-3%) and immunoprecipitates (IP, 40%) obtained with the indicated antibodies were resolved by SDS-PAGE. M indicates molecular mass in kDa. Western blots were probed with antibodies against Kir6.2 to detect Kir6.2 and Kir6.2-11HA (top panel), and against GFP to detect phogrin-EGFP (middle panel). (Bottom panel) Immunoprecipitates (40%) obtained with the indicated antibodies, probed with antibodies to 14-3-3 proteins. Note that the anti-14-3-3 ß antibody recognizes all 14-3-3 isoforms. Monomeric and dimeric species of 14-3-3 proteins are indicated (the dimer was only observed after crosslinking). The presence of an IgG signal in lanes 1 and 2 is because both anti-HA and anti-14-3-3 antibodies were of the same species (mouse). For anti-GFP IP a rabbit antibody was used, hence there is no IgG signal in lanes 3 and 4.

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