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Figure 7 | BMC Neuroscience

Figure 7

From: Brain-derived neurotrophic factor modulation of Kv1.3 channel is disregulated by adaptor proteins Grb10 and nShc

Figure 7

Grb10 protein-protein interactions with Kv1.3 causes decreased channel expression that require multiple Y residues. (A) HEK 293 cells were transiently transfected with cDNA encoding Kv1.3 (K) or Kv1.3 plus Grb10 (G) using either myc-tagged Kv1.3 (Upper panel) or wildtype Kv1.3 (Lower panel). Lysates were prepared and separated by SDS-PAGE as in Fig. 4. The Mr for Kv1.3 ranges from 55 to 72 kDa depending upon extent of phosphorylation (< Kv1.3). (B) Bar graph plot of the mean ± S.E.M. normalized immunodensity values of the Kv1.3 labeled band for experiments demonstrated in A and also transfection schemes including TrkB kinase (KT, KTG). Pixel density ratios (dashed line) were generated by normalizing to control K transfection condition within a single autoradiographic film to eliminate variability introduced by differential exposure times. * = significantly different compared with control K or KT transfection condition, respectively, by Student's t-test, arc sine transformation for percentage data, α ≤ 0.05. (C) Representative HEK 293 cells (of 30) transiently transfected with myc-tagged Kv1.3 (Myc) alone or plus Grb10 (Myc + Grb10). Channel protein was detected by incubation with anti-myc under non-permeabilizing conditions and then visualized with species-appropriate fluorescent-conjugated secondary antiserum. (D) (TOP) As in (A) using wildtype (K) or various Y to F point channel mutations or a W to F point channel mutation (W386F Kv1.3); transfection scheme was minus (-) or plus (+) Grb10 adaptor. The Mr for Kv1.3 ranges from 55 to 72 kDa depending upon extent of phosphorylation (< Kv1.3). (BOTTOM) As in (A) using Kv1.3 or two other Shaker subfamily members; transfection scheme was minus (-) or plus (+) Grb10 adaptor. NOTE: The Western analyses were performed in pairs for each channel member and therefore are not aligned to a single migration standard; Mr for Kv1.3 (55 to 72 kDa), Kv1.4 (74 kDa), Kv1.5 (64 kDa), and actin (42 kDa) varied. (E) Bar graph plot of the mean ± S.E.M. normalized immunodensity values for experiments demonstrated in (D, TOP). Pixel density ratios (dashed line) were generated as in Part (B) above, by normalizing to control transfection condition without Grb10 and comparing within a single autoradiographic film to eliminate variability introduced by differential exposure times. * = significantly different compared to minus Grb10 treatment by Student's t-test, Arc sine transformation for percentage data, α ≤ 0.05. (F) Bar graph plot of the mean ± S.E.M. normalized immunodensity values for experiments demonstrated in (D, BOTTOM). Analyses and statistical metric as in E.

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