Rabbit anti hdac3

The protein was extracted with lysis buffer (Beyotime, Shanghai, China) supplemented with 1 mM PMSF. After concentration determination, the protein underwent SDS-PAGE, and the gel concentration was set according to the protein molecule size. After electrophoresis, the protein in gel was transferred onto PVDF membrane (Abcam, Cambridge, MA, USA), which was blocked for 60 min to block the non-specific antigens, and incubated with following primary antibodies at 4 °C overnight: rabbit anti-SFMBT2 (1:1000; cat no. 25256-1-AP, Proteintech, Wuhan, Hubei, China), rabbit anti-HDAC3 (1:5000; cat no. ab32369, Abcam, Cambridge, MA, USA), mouse anti-SIAH1 (1:300; cat no. sc-81785, Santa Cruz, CA, USA), mouse anti-β-actin (1:1000; cat no. sc-47778, Santa Cruz, CA, USA). The PVDF membrane was rinsed with TBST, and incubated with goat anti-rabbit or anti-mouse IgG labeled with horse radish peroxidase (HRP) (1:5000; Beyotime, Shanghai, China) at 37 °C for 45 min. Finally, the membrane interacted with ECL reagent, and the signal exposure was performed in the dark. β-actin served as the internal control.

Pancreatic cancer cell lines (BxPC-3, Capan-1, SW1990, AsPC-1, HPAF-II, and HS-766T) and the normal human pancreatic ductal epithelial cell line, HPDE, were all obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). HS-766T cells were grown in 5% CO₂ with saturated humidity at 37 °C, cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mmol/L glutamine and 10% fetal bovine serum (FBS) (both from Gibco, Gaithersburg, MD, USA) and subcultured by harvesting with trypsin-EDTA. HPDE, BxPC-3, CAPAN-1, AsPC-1, and HPAF-II cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS. SW1990 cells were cultured in L-15 medium (Gibco) supplemented with 10% FBS. The 5-aza-2′-deoxycytidine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trichostatin A and 3-deazaneplanocin A (DZNeP) were purchased from Selleck Chemicals (Houston, TX, USA). Rabbit anti-Sp1, anti-EZH2, anti-Ago2, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-HDAC3 and anti-DNMT1 antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti-β-actin was purchased from Sigma-Aldrich.