Af2097

Chromatin immunoprecipitation (ChIP) was performed using a ChIP assay kit (Beyotime, Shanghai, China) and Dynabeads Protein G (Invitrogen) as described previously (Mehra and Wells, 2015 (link)). The qPCR primer sequences used are shown in Supplementary Table S5. Antibodies against Smad2 (ab71109; Abcam, Cambridge, England), Smad3 (ab28379; Abcam), Smad4 (AF2097, R&D), Meox1 (ab75895; Abcam), polymerase II (ab5095; Abcam), and immunoglobulin G (IgG) (ab2410; Abcam) were used for ChIP assays.

Total proteins extracted with the cell lysate buffer and protease inhibitors were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated with the primary antibody: HEATR1 (1 : 10000, ab241610, Abcam, CA, USA), FLAG (1 : 2000, F1804, Sigma, USA)， SMAD4 (1 : 1000, AF2097, R&D systems, MN, USA), and GAPDH (1 : 2000, sc-32233, Santa Cruz, Texas, USA) overnight after being blocked with 5% nonfat milk in TBS-T. Protein expression was detected with the Image System using Tanon 5200 (Tanon, Shanghai, China).