Western blotting of the Gli1, Gli3 and Tet1 proteins

Cells were incubated at 4°C for 30 minutes with a lysis buffer (10 mM Tris-HCl pH 7.4, 0.1% Triton X-100, 5 mM MgCl₂, 20 mM β-mercaptoethanol, 5 U/ml of DNase I, Protease Inhibitor Cocktail (Pierce) for Gli1 or Gli3; and 7M urea, 2M thiourea, 4% CHAPS, 5 mM PMSF, 1% DTT for Tet1). Total protein levels were determined using Bradford reagent for equal application on electrophoresis tracks. The protein samples were diluted in Laemmli buffer, subjected to SDS-PAGE (10% for Gli1 or Gli3 and 6% for Tet1), containing 0.1% SDS, and transferred to the PVDF membrane (Thermo Scientific). Immunoblotting was performed according to the Blue Dry Western protocol [32]. GAPDH was detected as a loading-control. Mouse monoclonal antibodies to Gli1 (Abnova) or GAPDH (Merck Millipore), at dilutions of 1:300 and 1:500, respectively, as well as rabbit polyclonal antibodies to Gli3 (Abcam) or Tet1 (GeneTex), at dilutions of 1:300 and 1:600, respectively, were used as primary antibodies. Horseradish peroxidase-conjugated goat antibodies against mouse or rabbit immunoglobulins from Sigma were used as secondary antibodies in dilutions of 1:2000 and 1:5000, respectively. Chemiluminescent detection of the protein bands was performed with Clarity Western ECL Blotting Substrate (Bio-Rad). Densitometry analysis was done by TotalLab quant software.