Ab12169

Piceatannol was purchased from Future Chem (Seoul, Korea). Anti-alpha smooth muscle actin (α-SMA; 1:1000, sc-130617), anti-CTGF (1:1000, sc-14939), anti-HDAC3 (1:1000, sc-11417), anti-HDAC4 (1:1000, sc-11418), anti-HDAC5 (1:1000, sc-133225), anti-TGF-β1 (1:1000, sc-146), anti-JNK (1:1000, sc-7345), anti-ERK1 (1:1000, sc-271269), and anti-GAPDH (1:1000, sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against collagen type I (1:1000, ab34710), HDAC2 (1:1000, ab12169), HDAC8 (1:1000, ab137474), and HDAC10 (1:1000, ab53096) were purchased from Abcam (Cambridge, MA, USA). Anti-fibronectin antibody (1:1000, MA5-11981) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-HDAC1 antibody (1:1000, 06–720) was purchased from Merck Millipore (Darmstadt, Germany). Anti-HDAC6 (1:1000, 7612), anti-Smad3 (1:1000, 9523), anti-Smad2 (1:1000, 3103), anti-Smad4 (1:1000, 9515), anti-p-Smad3 (1:1000, 9520), anti-p-JNK (1:1000, 9251), anti-p-p38 (1:1000, 4511), anti-p-ERK1/2 (1:1000, 4370), and anti-p38 (1:1000, 8690) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

ChIPs were performed for human ESCC cell lines (EC109 and TE-1) and were analyzed essentially according to the instructions of One-Day Chromatin Immunoprecipitation Kit (Millipore). The DNA precipitated by the target antibodies (DNMT1, Abcam, ab13537; HDAC2, Abcam, ab12169) was detected with qRT-PCR. The primer sequences of the ChIP-qPCR reaction were as follows: p16-1, 5′-CTG CTC TTA TAC CAG GCA ATG TA-3′ (sense) and 5′-CCT GTA CGA CTA GAA AGT GTC CC-3′ (antisense); p16-2, 5′-TTT CCC TAT GAC ACC AAA CAC C-3′ (sense) and 5′-CCG CGA TAC AAC CTT CCT AAC-3′ (antisense); p16-3, 5′-CCT CCT TGC GCT TGT TAT ACT CT-3′ (sense) and 5′-CCC TCC ACC ACC CTC ACT TA-3′ (antisense). Control PCRs for each antibody immunoprecipitation were performed using primers for GAPDH and IGFBP3 (DNMT1) or CD4 and von Willebrand factor (HDAC2) as negative and positive controls dependent on the antibody used. All PCR product sizes were confirmed by electrophoresis. Each ChIP experiment was done in triplicate and repeated at least three times.