nShc adaptor forms a protein-protein interaction with Kv1.3 channel. (A) HEK 293 cells were transiently transfected with cDNA encoding myc-tagged Kv1.3 (mK) plus TrkB (T) or nShc (S) or triply transfected with channel, kinase, adaptor combination using either wild-type (T) or mutant TrkB kinase (TShc-) lacking the Shc binding site. Forty-eight hour (h) post-transfection, cells were stimulated with 1 ng/ml BDNF (+) or vehicle control (-) for 10 minutes at 37°C. Five to 10 μg of whole-cell lysates from each respective transfection condition were separated by SDS-PAGE, electro-transferred to nitrocellulose, and probed with αShc (anti-Shc). Upper panel shows representative expression bands for three such experiments in which endogenous (lanes 1–4) versus transfected levels (lanes 5–10) of Shc protein are compared. In the lower panel, lysates were immunoprecipitated (IP) with an antiserum against the myc epitope to pull down Kv1.3 channel protein (anti-myc), separated by SDS-PAGE and nitrocellulose was probed with αShc (anti-Shc). Note direct co-IP of Shc and Kv1.3 (lanes 5–6) is independent of BDNF stimulation. Addition (lanes 7–8) and loss of TrkB Shc binding site (lanes 9–10) does not affect the co-IP. Lower panel expression bands are representative of three such experiments. The predicted molecular mass (Mr) for Shc is 46/52/66 kDa. (B) Similar experimental paradigm, protocol, notation, and sample size as in (A) but for Grb10 adaptor. Grb10 = G. Top paired panels show three repetitions in which myc-tagged Kv1.3 is transfected alone (mK) or including Grb10 (mKG). Lysates were probed with αGrb10 (anti-Grb10) to compare endogenous versus transfected levels of Grb10. IP of the channel (anti-myc) followed by probing with αGrb10 (anti-Grb10) fails to indicate any channel/adaptor complex. Bottom paired panels demonstrate that αGrb10 can effectively be immunoprecipitated (IP: Grb10, blot Grb10, lanes 7–8) but that it also does not co-IP with TrkB (lanes 9–10). Mr for Grb10 is 60 kDa.