Fig. S3

Validation of Tol2 vectors and establishment of heat shock inducible transgenic lines (A) Transient expression of Vangl2 FL protein by injecting Tol2 plasmid into Tg(hsp70:Gal4) embryos (i-iii). The 24 hpf embryos were heat shocked for 1 hr at 37°C and examined at 48 hpf. Control embryos contained the Tol2 plasmid but were not subjected to heat shock (i′-iii′). Embryos carrying the injected Tol2 plasmid express GFP in the heart (arrows in iii and iii′). Heat shocked embryos express RFP in the superficial cells of the yolk sac (i, arrow) and cells in the trunk (ii, arrow). RFP is not expressed in non-heat shocked embryos (i′, ii′). (B) Western blotting with anti-Myc antibody shows that the various Vangl2 variants are expressed in the Tol2 plasmid-injected embryos after heat shock (+). While most constructs are not activated in the absence of heat shock (-), there is a varying amount of weak (leaky) expression for N-PH, N-TM, PBMΔ, DTM, and C. A Western using anti-tubulin antibody was performed simultaneously to detect tubulin as a loading control. (C) Western blotting showing different levels of Vangl2 FL protein expression among four different stable transgenic lines. Lines A and D were selected for further analysis, and most experiments were performed with line D.