Examination of expression of Vangl2 and Celsr1 in chuzhoi mutants, and Ptk7 in Vangl2 and Celsr1 mutants. (A) Western blot analysis of Ptk7 expression in total cell lysates from E8.5 ScribCrc/Crc, Vangl2Lp/Lp, and Celsr1Crsh/Crshmutants, compared to heterozygous and wild-type littermates; fatty acid synthase (Fas) was used as a loading control. No obvious difference in Ptk7 expression levels was observed in any of the mutants. (B-D) Western blot analysis of Scrib (B), Vangl2 (C) and Celsr1 (D) expression in total cell lysates from E8.5 chuzhoi mutants, compared to heterozygous and wild-type littermates; fatty acid synthase (Fas) or β-tubulin were used as loading controls. Inclusion of protein extracts from circletail and loop-tail homozygotes were used to help validate anti-Scrib and anti-Vangl2 antibody specificity, respectively. No reproducible difference was observed between chuzhoi mutant and wild-type samples, for expression of either Scrib, Vangl2 or Celsr1. (E-K) Immunofluorescence on transverse sections of E8.0 embryos with antibodies for Ptk7 (E-G), Celsr1 (H, I) and Vangl2 (J, K) in wild-type (E, H, J), Celsr1Crsh/Crsh(F), Vangl2Lp/Lp(G) or chuzhoi mutant (I, K) embryos. Ptk7, Celsr1 and Vangl2 are detected around the membrane of neuroepithelial cells, and no clear expression difference was observed in the mutant embryos.