Anti dyrk1b

The protein of cells was extracted for immunoblotting as described previously [18 (link),19 (link),20 (link)]. Briefly, the cells were harvested with lysis buffer containing protease inhibitor cocktail. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes for further immunoblotting with the primary antibodies, including anti-DYRK1B (1:1000, 5672, Cell Signaling Technology), anti-His tag (1:1000, 12698, Cell Signaling Technology), and anti-ACTB (1:2000, A5441, Sigma) at 4 °C overnight, followed by probing with secondary antibody and ECL reagent. The membrane was scanned and analyzed for protein expression as uncropped images in Figure S1 with a BioSpectrum^® Imaging System (UVP, Upland, CA, USA).

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)