Figure 2.

CPE mediates activity-dependent TrkB membrane surface insertion in hippocampal neurons. A, PC12 cell were transiently transfected with Ctr siRNA or CPE siRNA. After 2 d, cells lysates were immunoblotted with CPE antibody to detect the CPE-knockdown efficiency. Endogenous β-tubulin was used as an expression control. B, Representative immunofluorescence images of TrkB surface levels in neurons (9 DIV) expressing the indicated constructs with or without Gly treatment (200 μm, 10 min). Surface TrkB levels were determined using ratiometric fluorescence assay. The siRNA constructs all expressed fused RFP, and we only captured images of RFP-positive neuron (pseudo blue color). The total TrkB receptors were fused with GFP (green color). The surface TrkB stained with M2 antibody followed by Cy5-conjugated goat anti-mouse antibody (pseudo red color). Anti-FLAG antibody labeled surface TrkB receptors, and the GFP fluorescence represented total receptor levels. Scale bar, 10 μm. C, Surface levels of TrkB receptor were analyzed as in B. Relative surface levels were normalized to that of TrkB-GFP (two-way ANOVA with Bonferroni's multiple comparisons test, F_(1,12) = 8.627, Ctr-Ctr siRNA vs Gly-Ctr siRNA **p = 0.0042; n = 4). D, Surface levels of TrkB and T1 receptors were evaluated by surface biotinylation assay in hippocampal neurons transfected with the indicated siRNA. Surface labeled TrKB were detected by streptavidin pull-down followed by anti-TrkB immunoblotting. E, Quantification of the surface levels of TrkB receptors by the ratio of surface to total TrkB intensity normalized to the control group (two-way ANOVA with Bonferroni's multiple comparisons test, F_(1,12) = 7.75, Ctr-FAM-Con VS Gly-FAM-CPE siRNA **p = 0.0019; n = 4). F, Quantification of the total TrkB receptors normalized to the control group. Error bars indicate ± SEM. G, Quantification of the surface levels of T1 receptors by the ratio of surface to total T1 intensity normalized to the control group. H, Representative epifluorescence images from the TrkB internalization assay. The Control, Ctr siRNA, and CPE siRNA constructs all express nonfused GFP, and we only captured images of GFP-positive neurons (GFP image not shown). The total pool of Flag-TrkB initially present at the cell surface was labeled with 594-M2 (Internal + surface group, green, pseudo color). After BDNF treatment at 37°C for 15 min, neurons were fixed by 4% PFA under nonpermeabilizing conditions, and the receptors remaining at the cell surface were labeled with Cy5-conjugated secondary antibody (surface group, red, pseudo color). Scale bar, 10 μm. I, Quantitation of internalized TrkB levels in H was performed. All of the data are presented as mean ± SEM determined from analysis of more than three independent experiments (n ≥ 25 cells for each condition per experiment).