Fig. 2
From: Regulation of glypican 6-mediated Wnt activation maintains TDP-43 nuclear localization in neurons
GPC6 potentiates Wnt7b signaling transduction. (a,b) Representative western blots (a) of SH-SY5Y cells transfected with plasmids expressing HA-GPC6 and 250ng Wnt7b (see Fig. S2c) and graph (b) quantifying non-p-β-catenin levels normalized to Tubulin. Arrow marks full-length HA-GPC6; Asterisk marks N-terminal HA-GPC6 protein after cleavage by furin convertase. GFP-GFP vs. GFP-GPC6: ns = 0.9449; Wnt7b-GFP vs. Wnt7b-GPC6: *p = 0.0253; GFP-GFP vs. Wnt7b-GFP: ns = 0.7818. RM two-way ANOVA. n = 3, mean ± s.e.m. (c). Graph quantifying GPC6 mRNA levels treated with siRNAs. GFP-scramble vs. GFP-siGPC6: ***p = 0.0003; Wnt7b-scramble vs. Wnt7b-siGPC6: ***p = 0.0003. RM two-way ANOVA with Sidak’s multiple comparison test. n = 4, mean ± s.e.m. (d). Representative western blots of SH-SY5Y cells transfected with 500ng of plasmids expressing Wnt7b (see Fig. S2c) and treated with siRNAs. e. Graph quantifying non-p-β-catenin protein levels normalized to Tubulin. GFP-scramble vs. GFP-siGPC6: ns = 0.8438; GFP-scramble vs. Wnt7b-scramble: *p = 0.0372; Wnt7b-scramble vs. Wnt7b-siGPC6: *p = 0.0338; GFP-siGPC6 vs. Wnt7b-siGPC6: ns = 0.6939. RM two-way ANOVA. n = 4, mean ± s.e.m. (f,j). Topflash luciferase assay in STF cells quantifying Firefly luciferase activity, reporting Wnt transcriptional activity, normalized to Renilla expressed under a universal promoter. (f) GFP vs. Wnt7b with Fzd1: ***p = 0.0003; Fzd3: ns p = 0.9898; Fzd4: *p = 0.0207; Fzd7: **p = 0.0086; Fzd8: ****p < 0.0001. Ordinary two-way ANOVA with Sidak’s multiple comparison test, with a single pooled variance. n = 5, mean ± s.e.m. (g) Fzd1: GFP-GFP vs. GFP-GPC6 **p = 0.0034; GFP-GFP vs. GFP-Wnt7b ****p < 0.0001; Wnt7b-GFP vs. Wnt7b-GPC6 **p = 0.0019; GFP-GPC6 vs. Wnt7b-GPC6: ****p < 0.0001. (h) Fzd4: GFP-GFP vs. GFP-GPC6 ns p = 0.1370; GFP-GFP vs. GFP-Wnt7b **p = 0.0076; Wnt7b-GFP vs. Wnt7b-GPC6 *p = 0.0358; GFP-GPC6 vs. Wnt7b-GPC6: **p = 0.0034. i: Fzd7: GFP-GFP vs. GFP-GPC6 ***p = 0.0008; GFP-GFP vs. GFP-Wnt7b ****p < 0.0001; Wnt7b-GFP vs. Wnt7b-GPC6 **p = 0.0020; GFP-GPC6 vs. Wnt7b-GPC6: ***p = 0.0002. j: Fzd8: GFP-GFP vs. GFP-GPC6 ns p = 0.9327; GFP-GFP vs. GFP-Wnt7b ***p = 0.0004; Wnt7b-GFP vs. Wnt7b-GPC6 ns p = 0.0644; GFP-GPC6 vs. Wnt7b-GPC6: ***p = 0.0002. RM-two-way ANOVA. n = 5, mean ± s.e.m.
