Gsk 3β

Protein of cells cultured in specific media was extracted by RIPA buffer (Invitrogen), separated by 12% SDS-PAGE gels, and transferred onto PVDF membrane. The blots were blocked with 5% non-fat dried milk, incubated with primary antibodies including β-catenin (Beyotime, AC106), GSK-3β (Beyotime, AG751), p-GSK-3β (Beyotime, AF1531), and GAPDH (Beyotime, AF0006) at 4 °C overnight, and with secondary antibodies at 37 °C for 2 h. The immunoreactive bonds were developed by ECL kit (solarbio, PE0010) and exposured by the ImageQuant LAS 4000 system.

Tetrahydrocurcumin was purchased from Yuanye Biotechnology Co. Ltd (Shanghai,China). E-cadherin, ZEB, Snail, Twist, HIF-1α, p-mTOR, mTOR, p-ERK, ERK, GAPDH and β-actin antibodies were purchased from Santa cruz Biotechnology (Santa cruz, CA, USA). Vimentin, N-cadherin, VEGF, MMP-2, MMP-9, LaminA/C, Atg5, Atg7 and Beclin-1 antibodies were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). LY294002, SB203580, Rapamycin, β-catenin, p-Akt, Akt, p-JNK, JNK, p-p38, p38, IκB-α, p65, Hsp90 and GSK-3β antibodies were obtained from Beyotime Institute of Biotechnology (Haimen, China). Antibody against LC3B was purchased from Abcam (Cambridge, UK). All other reagents were from common commercial sources.

Hep3B cells were seeded in 6‐well plates at 2 × 10⁵ cells/well and treated with 10 mg/ml HP for 0, 1, 3, 6, and 12 hr. Cell protein lysates were separated on 12% SDS‐PAGE (sodium dodecyl sulfate–polyacrylamide gels) and transferred onto nitrocellulose membranes (Millipore). Membranes were blotted with primary antibodies against caspase‐3 (Cell Signaling Technology, #9661), Bad (Cell Signaling Technology, #9268), Bcl‐2 (Cell Signaling Technology, #15071), Akt (Cell Signaling Technology, #2920), p‐Akt (Cell Signaling Technology, #4060), Gsk‐3β (Beyotime, #AG751), p‐Gsk‐3β (Beyotime, #AG753), β‐catenin (Beyotime, #AC106), p‐β‐catenin (Bioss, #bs‐3085), and β‐actin (Cell Signaling Technology, #3700) (dilution 1:2,000) at 4°C overnight. Membranes were washed five times with Tris‐buffered saline containing Tween‐20 (TBST) (10 mM Tris‐HCl (pH 7.5), 150 mM NaCl, and 0.2% Tween‐20) and were subsequently incubated with Horseradish Peroxidase‐conjugated goat antirabbit IgG (Sangon Biotech) or antimouse IgG (Sangon Biotech) for 1 hr at room temperature (RT). After the removal of excess antibodies by washing with TBST, signal detection was performed using chemiluminescence (GE Healthcare Life Sciences) according to the manufacturer's instructions.