Figure 1: Impaired TrkB trafficking to the cell surface at synapses in ARHGAP33 KO mice.
(a) Protein structure of a brain-enriched SNX protein, ARHGAP33. ARHGAP33 has an N-terminal PX domain, an SH3 domain and a RhoGAP domain. (b,c) Decreased cell-surface expression of TrkB in ARHGAP33 KO mice. Biotinylated cell-surface proteins (upper) and total lysates (lower) of WT and ARHGAP33 KO neurons (14 DIV) were immunoblotted with anti-TrkB, anti-TrkC, anti-SORT1, anti-GAPDH and anti-ARHGAP33 antibodies. (b) Representative blots. (c) Quantification of surface expression (each, n=8; TrkB, P=7.8 × 10−4; TrkC and SORT1, P>0.05; Mann–Whitney U-test). The expression levels of TrkB, TrkC and SORT1 in ARHGAP33 KO neurons were normalized to those in WT neurons (The averaged WT values were set to 100%). (d,e) Decreased TrkB in the isolated PSD fraction of ARHGAP33 KO mice. The isolated PSD fraction and total lysates of WT and ARHGAP33 KO mice were immunoblotted with anti-TrkB, anti-PSD-95, anti-SORT1, and anti-ARHGAP33 antibodies. Representative blots (d). Quantification for the isolated PSD fraction (each, n=8, TrkB, P=7.7 × 10−4; SORT1 and PSD-95, P>0.05; Mann–Whitney U-test) and for the total lysate (each, n=8, P>0.05; Mann–Whitney U-test; e) The expression levels of TrkB, SORT1 and PSD-95 in the PSD fraction and total lysate from ARHGAP33 KO mice were normalized to those from WT mice (The averaged WT values were set to 100%). Note that the amounts of PSD-95 and SORT1 in the isolated PSD fraction from ARHGAP33 KO mice were not significantly different from those in the fraction from WT mice. *P<0.05. NS, not significant. Bars show median values. All western blots show representative results from eight independent experiments performed using different mice.
