Figure 7: Cooperative facilitation of TrkB trafficking by ARHGAP33 and SORT1.

(a,b) Increased TrkB surface expression by ARHGAP33 and SORT1. HEK293T cells were transfected with TrkB, ARHGAP33 and SORT1 as indicated in b (1–4). Representative data from eight independent experiments of immunostaining of surface TrkB in HEK293T cells with an antibody against the extracellular region of TrkB (a). Scale bar, 10 μm. Representative blots (left) and the quantification of the surface TrkB (right) (b). Biotinylated cell-surface proteins were immunoblotted with the indicated antibodies. The surface expression of TrkB was enhanced by simultaneous expression of ARHGAP33 and SORT1 compared with the expression of ARHGAP33 alone (each n=8; ARHGAP33 alone versus ARHGAP33 and SORT1, P=0.004, Kruskal–Wallis test followed by post hoc Steel–Dwass tests). Western blots show representative results from eight independent experiments. The averaged value of surface TrkB level in cells expressing ARHGAP33 alone (lane 3) was set to 100%. *P<0.05. Bars show median values. (c) Requirement of SORT1 in ARHGAP33-mediated TrkB trafficking. Biotinylated cell-surface proteins were immunoblotted with the indicated antibodies. Representative blots (left), quantification of surface TrkB expression (centre) and quantification of SORT1 expression (confirmation of SORT1 knockdown; right; each n=10; surface TrkB, WT versus ARHGAP33 KO in control neurons, corrected P=6.0 × 10⁻⁴; WT versus ARHGAP33 KO in SORT1 knockdown neurons, corrected P>0.05; ARHGAP33 KO versus ARHGAP33 KO plus SORT1 knockdown, corrected P>0.05, Mann–Whitney U-test with the Ryan’s correction). Western blots show representative results from 10 independent experiments performed using neurons from different mice. The averaged values of WT mice in the control neurons were set to 100%. *P<0.05; cont., control; KD, knockdown; NS, not significant. Bars show median values. Note that the MISSION shRNA construct (TRCN0000034496) was used. (d) Strongly correlated expression of SORT1 and ARHGAP33 in immortalized lymphocytes from human blood (r=0.42, P<0.001, Spearman’s rank order correlation test). Quantitative RT-PCR analysis of ARHGAP33 and SORT1 expression in immortalized lymphocytes was performed. Then, the levels of SORT1 and ARHGAP33 expression in each sample were plotted.