Figure 8

Kv1.3 channel co-transfection suppresses receptor tyrosine kinase (RTK) expression and concomitant phosphorylation. (A) HEK 293 cells were transiently transfected with cDNA encoding TrkB kinase (T) or Insulin receptor kinase (I) minus or plus Kv1.3 channel (KT, KI). Cells were stimulated with vehicle (-) vs. BDNF or insulin (+), and lysates were prepared and separated by SDS-PAGE as in Fig. 4. Upper panel lysates were IP with antiserum that recognizes tyrosine phosphorylated proteins (anti-4G10) and then were blotted with either αTrkB or αIR to quantify the degree of kinase phosphorylation (pIR or pTrkB). Lower panel shows representative expression bands for three such experiments in which nitrocellulose was probed with either αTrkB or αIR to recognize the respective RTK. (B) Bar graph of the mean ± S.E.M. normalized immunodensity values for either TrkB or IR kinase expression in the presence (striped bar) or absence (solid bar) of Kv1.3 co-expression under unstimulated or hormone/trophic factor stimulation (+ BDNF or + Insulin), respectively. Pixel density ratios (dashed line) were generated by normalizing kinase expression levels with the channel (KT or KI) to that of control transfection condition without Kv1.3 (T or I) and comparing within a single autoradiographic film to eliminate variability introduced by differential exposure times. * = significantly different compared to kinase alone transfection condition by Student's t-test, Arc sine transformation for percentage data, α ≤ 0.05.