β actin sc 7963

DMEM and F12 medium, fetal bovine serum (FBS), trypan blue, penicillin G, and streptomycin were obtained from Invitrogen (Gaithersburg, MD, USA). Dimethyl sulphoxide (DMSO), ribonuclease A (RNase A), and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against JNK, p38 (sc-7149), p-p38 (Tyr¹⁸², sc-7973), ERK, p-ERK (Tyr²⁰⁴, sc-7976), COX-2, and β-actin (sc-7963) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against SP-1 (5407-S) was purchased from Epitomics. Antibody against p-JNK (Thr¹⁸³/Tyr¹⁸⁵, #07-175) was purchased from Millipore. Anti-rabbit, anti-goat and anti-mouse IgG peroxidase-conjugated secondary antibodies were purchased from Pierce (Rockford, IL, USA). Annexin V-FITC staining kit was purchased from Strong Biotech Co. Ltd. (Taipei, Taiwan).

Total protein extraction was carried out with a RIPA lysis buffer (Beyotime Biotechnology). The protein concentration was determined by the BCA Protein Assay Kit (Pierce Biotechnology). Equivalent proteins were electrophoresed on 10% SDS‐PAGE gel and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. Thereafter, membranes were blocked in 5% skim milk, followed by incubation with primary antibody at 4°C overnight, probed by appropriate secondary antibody at room temperature for 1 hour and then visualized with chemiluminescence molecular imaging system (Bio‐Rad). The following primary antibodies were applied: anti‐USF1 ((ab125020; Abcam), anti‐E‐cadherin (sc‐8426; Santa Cruz), anti‐vimentin (sc‐6260; Santa Cruz), β‐actin (sc‐7963; Santa Cruz), anti‐Nanog (ab109250; Abcam), anti‐SOX2 (ab137385; Abcam) and anti‐OCT4 (ab184665; Abcam). β‐actin served as the loading control.