Fig. 4

a Representative immunoblot of 48 hpf zebrafish embryos. Twenty-four hpf embryos were incubated with 100 µM cyclopamine or with vehicle alone (4 embryos per point) for 24 h and the expression of GlyT2 was analyzed by immunoblotting. GAPDH is used as protein loading control. b Quantification of GlyT2 expression is normalized to the corrected signal against GAPDH. **p (Veh vs CYC 100 μM) = 0.0014, using Kolmogorov–Smirnov test, n = 8. c Relative mRNA levels of GlyT2 in 24 hpf zebrafish embryos treated with 100 µM cyclopamine for 24 h were determined by qPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene (arbitrary units). n.s. = not significantly different using Mann–Whitney U test, n = 4. CYC cyclopamine. d Dechorionated 48 hpf zebrafish embryos treated as in a. were fixed in 4% PFA and embedded in OCT cryostat embedding medium. Ten micrometer spinal cord transversal sections were incubated with anti-GlyT2 antibody (green) and DAPI (blue). e Quantification of GlyT2 fluorescence intensity. ****p < 0.0001, using Mann–Whitney U test, n (Veh) = 29, n (CYC) = 30. CYC cyclopamine, D dorsal, V ventral.