Supplementary Figure 15: Western blot of Wif1 and Cldn5 protein levels in endothelial culture treatments.

(a-b) Wif1 protein levels were measured in primary cultured OPCs from APCfl/fl control and Olig2-cre:APCfl/fl littermate mice using Western blot (a) and quantified in (b)(n=4 independent experiments; APCfl/fl vs. Olig2-cre:APCfl/fl *p = 0.0286). Data were analyzed by unpaired two-sided Student’s t test. (c-d) Primary cultured endothelial cells were treated with recombinant Wif1 protein, Wnt3a or Wif1+Wnt3a. Claudin5 (Cldn5) protein levels in treated endothelial cells were measured using Western blot (c). Quantification of these Cldn5 protein levels in treatment groups are shown in (d)(n=4 independent experiments; PBS vs. Wif1 **p = 0.0027; PBS vs. Wnt3a ****p = 4.19 E-7; Wnt3a vs. Wif1+Wnt3a ####p = 6.71 E-8). Data were analyzed by one-way ANOVA. (e-f) Cldn5 protein levels were measured by western blot in primary endothelial cultures treated for 2 days with conditioned medium from APCfl/fl or Olig2cre:APC fl/fl OPCs (labelled ‘WT-CM+BSA’ and ‘Olig2cre:APC fl/fl-CM+BSA’ respectively) or conditioned medium from the same OPCs depleted of Wif1 protein by overnight anti-Wif1 antibody (+Ab) treatment and agarose bead pull down. Quantification of Cldn5 protein levels in these treatment groups are shown in (f)(n = 4 independent experiments; WT-CM+BSA vs. APC-CM+BSA ***p = 0.0005; APC-CM+BSA vs. APC-CM+Ab #p = 0.0158). Data were analyzed by one-way ANOVA. (g) Wif1+ mRNA expressing cell density per mm² in P9 Olig2cre:APCfl/fl mouse spinal cord versus APCfl/fl controls (n = 4 animals; APCfl/fl vs. Olig2-cre:APCfl/fl ****p = 2.67 E-5). Data were analyzed by unpaired two-sided Student’s t test. Values are mean ± s.d.