FIGURE 4.

KCTD12 stably interacts with GABA_B receptors during receptor activation in transfected COS-1 cells. A, co-immunoprecipitation of KCTD12 with surface GABA_B receptors from cells expressing Myc-GABA_B1 (Myc-GB1), HA-GABA_B2 (HA-GB2), and FLAG-KCTD12 (Flag-KCTD12) with and without receptor activation with baclofen (100 μm, 15 min). The amount of KCTD12 protein that co-immunoprecipitated with GABA_B receptors was similar in the presence and absence of baclofen. The absence of α-actin in the immunoprecipitates (IP) supports that surface GABA_B receptors were selectively isolated. Data are representative of 3 independent experiments done in duplicates. B, BRET donor saturation curves were generated in cells expressing fixed amounts of Myc-GABA_B2, Rluc-KCTD12, or Rluc-KCTD10 and increasing amounts of GABA_B1-YFP (GB1-YFP). Cells were preincubated with 100 μm baclofen for 15 min (filled circles and squares) or with PBS only (open circles and squares). BRET is expressed as milliBRET units (mBU) determined as net BRET × 1000. The results are the mean ± S.D. of 3–4 individual saturation experiments. The curves were fitted using a nonlinear regression equation assuming a single binding site (GraphPad Prism).