Figure 8. Yip1A, Rab6, or Kif5B depletions disturb 5-HT1AR targeting toward distal dendrites. A–E, Immunofluorescence of neurons transfected with the 5-HT1AR-eGFP alone (A) or cotransfected with the 5-HT1AR-eGFP plus siRNAs against endogenous Yip1A (B), endogenous Rab6 (C), endogenous Kif5B (D), or control siRNA (E). Immunolabeling was performed with anti-GFP antibody to enhance the GFP signal (green, left) or anti-α-tubulin antibody (red, middle). Overlay and zoom on the soma are shown on the right. On the right panels, the graphs represents the cumulated fluorescence intensities of the 5-HT1AR (cumulated fluorescence intensity, arbitrary unit) along the longest dendrite of monitored neurons (μm), in control conditions (blue) compared with experimental conditions (red) with siRNA against Yip1A, Rab6 or Kif5B. Scale bars, 50 μm. F, Expression level of the 5-HT1AR-eGFP was similar in siRNA-transfected neurons in comparison with control conditions after visualization on Western blot of protein extracts 48 h after transfection of neurons at DIV7. G, Downregulation of the Yip1A, Rab6, and Kif5B proteins by siRNAs in primary cultures of rat hippocampal neurons 48 h after transfection of neurons at DIV7. Yip1A (G1), Rab6 (G2), and Kif5B (G3) were detected by Western blots of protein extracts; α-tubulin was immunolabeled in the same membrane to normalize the amount of extract. All neurons were cotransfected with 5-HT1AR-eGFP alone (Control) or with 5-HT1AR-eGFP plus siRNA directed against endogenous Yip1A, Rab6, or Kif5B, or control siRNA. H, Normalized average 5-HT1AR fluorescence in dendrites along 50–100 μm from soma. Mean fluorescence at 50 μm was calculated for 20 neurons for each condition and then normalized to the mean value of the control condition [siRNA(control), siRNA(Yip1A), siRNA(Rab6), and siRNA(Kif5B)]. The fluorescence of 5-HT1AR is significantly lower in dendrites after siRNA-induced downregulation of Kif5B, Rab6, or Yip1A. N = 20–30 neurons per condition. Bar graphs show mean + SEM. *p < 0.05, **p < 0.01 ***p < 0.001 (one-way ANOVA with Dunnett's multiple comparison post hoc test).