Anti gli2 h 300

Cells were lysed using RIPA buffer (Tris-HCl pH 7.6 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, sodium pyrophosphate 2 mM and protease inhibitors). Lysates were separated on 8% acrylamide gel and immunoblotted using standard procedures. The following antibodies were used: anti-Arrb1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-Nanog (Cosmo Bio Co, Japan), anti-Actin I-19 (sc-1616; Santa Cruz Biotechnology), anti-β-III-Tubulin (MAB 1637 Millipore), anti-Gli1 H-300 (sc-20,687; Santa Cruz Biotechnology), anti-acetyl-Gli1 (Lys518) (Eurogentec) [32 (link)], anti-p300 C-20 (sc-585; Santa Cruz Biotechnology), anti-FLAG M2-Peroxidase (HRP) (A8592 Sigma), anti-HA (sc-7392 Santa Cruz), anti-Gli2 H-300 (sc-28,674; Santa Cruz Biotechnology), anti-Smo N-19 (sc-6366; Santa Cruz Biotechnology), anti-Sox2 (MAB4343 Millipore). HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) were used in combination with enhanced chemo-luminescence (ECL Amersham).
For immunoprecipitation assay antibody sources and concentrations used were: Protein G Plus-Agarose (sc-2002; Santa Cruz Biotechnology); anti-FLAG M2 Affinity Gel (Sigma A2220, IP 30⌠l), anti-FLAG M2-Peroxidase (HRP) (A8592 Sigma, western blotting 1:5000), anti-HA (sc-7392 Santa Cruz, 1:1000); anti-myc-HRP.

Total RNA was isolated using TRI-reagent (Molecular Research Center Inc., Cincinnati, OH, USA) followed by LiCl purification. Precipitated and purified RNA was used for cDNA synthesis using Superscript II reverse transcriptase (Life Technologies, Thermo Fisher Scientific) according to the manufacturer's instructions. qPCR was done on a Rotorgene Q (Qiagen, Venlo, Netherlands) using GoTaq qPCR Mastermix reagent (Promega, Fitchburg, WI, USA). HH target genes were identified with primers as described in [16 (link)].
For Western blot analysis, proteins were visualized with horseradish peroxidase-conjugated secondary antibodies in combination with enhanced chemiluminescence detection system (GE Health Care, Chalfont St Giles, United Kingdom). The following antibodies were used: anti-GLI1 (V812; Cell Signaling Technology, Danvers, MA, USA), anti-GLI2 (H-300, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GLI3 (R&D Systems, Minneapolis, MN, USA), anti-DYRK1A, anti-DYRK1B (Cell Signaling Technology), anti-SUFU (C-15, Santa Cruz Biotechnology), anti-STAT3 (BD Biosciences, San Jose, CA, USA), anti-STAT5 (3H7, Cell Signaling Technology), anti-Beta Catenin (Cell Signaling Technology), anti-ACTB (Santa Cruz Biotechnology)