4.4. Immunoprecipitation and Coomassie Staining

For immunoprecipitation (IP), proteins were extracted from GANT61-treated FaDu and A253 cell lines using TENN buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, pH 8.0) supplemented with protease inhibitors (Roche, Basel, Switzerland). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Immunoprecipitation was performed using Protein G-coated Dynabeads (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions (Invitrogen, Rev. 005, Carlsbad, CA, USA). For Gli3 immunoprecipitation, 1000 μg of proteins and 5 μg of Gli3 antibody (AF-3690, R&D Systems, Minneapolis, MN, USA) were used for each IP sample. Samples were incubated with the Dynabead–antibody complex overnight at +4 °C. Samples were eluted with 1× loading buffer and heated for 5 min at 95 °C before electrophoresis in 7% PAA gel. After gel electrophoresis, the proteins were fixed in 50% (v/v) ethanol and 10% (v/v) acetic acid for 1 h at room temperature followed by further fixation in 50% (v/v) methanol and 10% (v/v) acetic acid overnight at +4 °C. The gels were stained in Coomassie Blue R250 (LKB Bromma, Bromma, Sweden) staining solution (3g/L Coomassie Blue R250, 45% (v/v) methanol, 10% (v/v) acetic acid in water) for 4 h with gentle agitation, and afterwards destained in 50% (v/v) methanol and 10% (v/v) acetic acid. The destaining solution was changed several times until the protein bands were completely visible. The gels were stored in 5% (v/v) acetic acid at +4 °C.