Fig. 6.

Coexpression of Nedd4-2 with Kv1.3 causes a decreased expression of the channel, which can be relieved with the addition of Grb10 adapter protein. A, Western blot of a homogeneous front of cell lysates (Lysates) prepared from HEK 293 cells transfected with Kv1.3 channel (K). Proteins were separated by 10% SDS-PAGE and electrotransferred to nitrocellulose. Nitrocellulose blots were probed with antiserum generated against the Kv1.3 COOH terminus (see text, anti-FSU120) at the noted dilutions ranging from 1:800 to 1:10,000. M_r = 58 kDa for the Kv1.3 channel (Kv1.3), also visible are the high molecular weight multimers of the 4 channel subunits (HMW Kv1.3i, > 200 kDa). B: same separation strategy as in A, but where individually transfected samples are first immunoprecipitated (IP) with anti-myc (1:400), separated by SDS-PAGE, and then blotted with anti-FSU-120 (1:1,000). C: same separation strategy as in A, but lysates are probed for anti-myc (1:400) or anti-actin (1:800). Bars above Western blot indicate repeated transfection plan of 2 different trial sets. M, Myc-tagged Kv1.3 channel; N = Nedd4-2; G, Grb10; D, dead Nedd4-2 (Nedd4-2CS). D: bar graph of the normalized immunodensity values of the collective protein bands and correlate sample size for the noted transfection conditions. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, one-way ANOVA with SNK post hoc test; n = number of transfection samples. Dashed line, immunodensity value for Kv1.3 transfected condition alone.