Anti rabbit gli1 ab

For Western blotting, cells were lysed with RIPA buffer (150 mM NaCl, 50 mM Tris base pH 8.0, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM DTT, 1 mM PMSF, and 1 mM Na₃VO₄) supplemented with Complete Protease Inhibitor Tablets (Roche) and phosphatase inhibitor (Sigma). Proteins were separated on a 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) followed by transfer (220 mA for 1 hour) to an Immobilon-P membrane (Millipore). The membrane was incubated at 4°C overnight in 5% skim milk in TBST (Tris Buffered Saline with Tween 20) with anti-rabbit GLI1 Ab (#2553, Cell Signaling) or anti-rabbit S6K1 Ab (sc-230, Santa Cruz Biotechnology) followed by incubation with goat anti-rabbit secondary antibodies for 1 hour in 5% skim milk in TBST and visualized using chemiluminescent substrate (Thermo Scientific). The Western blot experiments were done at least in triplicate and representative experiments are shown.

For Western blot analysis, cells were lysed with RIPA buffer (150 mM NaCl, 50 mM Tris base pH 8.0, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM DTT, 1 mM PMSF, and 1 mM Na₃VO₄) supplemented with Complete Protease Inhibitor Tablets (Roche) and Phosphatase Inhibitor Cocktail 3 (Sigma). Proteins were separated on a 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) followed by transfer (220 mA for 1 hour) to an Immobilon-P membrane (Millipore). The membrane was incubated at 4°C overnight in StartingBlock™ T20 (TBS) Blocking Buffer (#37543, Thermo Scientific) with monoclonal anti-mouse β-Actin antibody (A5441, Sigma-Aldrich), anti-rabbit GLI1 Ab (#2553, Cell Signaling Technology) or ERα antibody (sc-543, Santa Cruz Biotechnology), followed by incubation with goat anti-rabbit or anti-mouse secondary antibodies for 1 hour in StartingBlock™ T20 (TBS) Blocking Buffer and visualized using Pierce ECL chemiluminescent substrate (Thermo Scientific).