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. 2019 Sep 12;116(43):21563–21572. doi: 10.1073/pnas.1902790116

Fig. 1.

Fig. 1.

Expression, validation, and membrane fluorolabeling of human p75NTR constructs. (A) Scheme of the S6-p75NTR construct. The S6 tag is inserted between the signal peptide (SP) and cysteine-rich domains 1 to 4 (CRD1-4). CD, chopper; DD, death domain. (B) S6-p75NTR–EGFP (green) localizes in the plasma membrane and, to a minor extent, on the nuclear membrane in SHSY5Y cells (Left) as endogenous p75NTR (green, Top Right ) in PC12 cells expressing farnesyl (farn)-GFP or lamin-RFP (both red, Right). (Bottom Right) Intensity (I) vs. distance (d) plot along yellow dashed lines. (Scale bar, 10 μm.) (C) S6-p75NTR is palmitoylated. (Top) Scheme of the hydroxylamine (HA)-catalyzed palmitoyl/biotin exchange. (Bottom) Western blot showing the streptavidin pulldown (Pd) and the corresponding nonpalmitoylated/biotinylated supernatant (Spn); the control reaction without HA is also shown. WCE, whole cell extract. (D) S6-p75NTR is polarized in developing hippocampal neurons. The maximum intensity projection of a TIRF movie of Qdot-labeled S6-p75NTR (white) and the area explored by S6-p75NTR (blue) superimposed on the cell mask (gray) are shown. DIC, differential interference contrast. (Scale bars, 5 μm.) The graph shows the relative enrichment in the explored area in somas and neurites. ***P < 0.001, paired Student’s t test (2-tailed). Bars are mean ± SEM. The dotted red line is the value expected for a nonpolarized localization. (E, Left) Confocal images of growth cones of hippocampal neurons transfected with TagRFP-actin only (red, Top) or with S6-p75NTR (green) and TagRFP-actin (Bottom), untreated or after 30 min of incubation with 20 ng/mL proNGF. (Scale bars, 5 μm.) (E, Right) Quantification of the growth cone area is reported in the graph. Bars are mean ± SEM. ***P < 0.001, 1-way ANOVA (Bonferroni multiple comparisons). nt, nontransfected. (F, Top) Cleaved-caspase-3 (red)/MAP2 (green) immunofluorescence images to quantify proBDNF-induced apoptosis in cortical neurons from wt and p75 KO mice (DAPI is shown in blue). (Scale bar, 20 μm.) (F, Bottom) Percentage of cleaved caspase-3–positive neurons is reported as mean ± SEM in the graph. ***P < 0.001, unpaired Student’s t test (2-tailed); ###P < 0.001, 1-way ANOVA (Tukey’s multiple comparisons). ns, not significant at the 0.05 level. nt, nontransduced.