Pre cast 4 15 polyacrylamide gels

Fst-expressing somite tissue from wild-type chick embryos was dissected and cultured in Dulbecco's modified Eagle medium, 10% foetal bovine serum and 1% penicillin/streptomycin for 4 h before transfecting with 1 mM translation-blocking FstMO (Gene Tools; sequence 5′-GATCCTCTGATTTAACATCCTCAGC-3′) or 1 mM StdMO negative control (Gene Tools; sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′) using Endoporter PEG (Gene Tools). Protein was extracted after 48 h. Protein lysate (30 μg) was run on pre-cast 4-15% polyacrylamide gels (Bio-Rad) and blotted onto polyvinylidene fluoride membrane (Bio-Rad). Primary antibody against Fst (Abcam ab47941; 1:2000) was applied at 4°C overnight and secondary polyclonal goat anti-rabbit-HRP (Cell Signaling Technology, 7074; 1:2000) was applied for 1 h at room temperature. Primary antibody against HSC70 (Santa Cruz, sc-7298; 1:2500) was applied at 4°C overnight and secondary polyclonal goat anti-mouse-HRP (Agilent, P0447; 1:1000) was applied for 1 h at room temperature. The blots were treated with an ECL substrate kit and imaged.

Somites of wild-type embryos were dissected and cultured in Dulbecco's modified Eagle medium, 10% fetal bovine serum, 1% penicillin/streptomycin for 4 h before being transfected with Gli3 MO (1 mM) or control MO (1 mM) using Endoporter PEG (Gene Tools) and protein extracted after 48 h. Somites from AM133- or AMscr-injected embryos were dissected for protein extraction. Protein lysate (31.5 μg) was run on pre-cast 4-15% polyacrylamide gels (Bio-Rad) and blotted onto polyvinylidene fluoride membrane (Bio-Rad). Primary antibody against Gli3 (1:200, 6F5 Gli3N, Genentech, Wen et al., 2010 (link)) was applied at 4°C overnight before secondary polyclonal goat anti-mouse HRP (1:1000, P0447, DAKO) was applied for 1 h at room temperature. The blots were treated with an ECL substrate kit (GE Healthcare) and imaged. Primary antibody against actin (1:1000, ab3280, Abcam) was applied at 4°C overnight; secondary polyclonal goat anti-mouse HRP was applied for 1 h at room temperature. The blots were treated with an ECL substrate kit and imaged. Quantification of blots was performed using ImageJ.