Enhanced chemiluminescence reagent

The cell lysates were prepared with ice-cold RIPA buffer and centrifuged (12000g for 15 min at 4℃). The protein concentration was determined by BCA assay. 30μg protein sample was electrophoresed on a 10% SDS polyacrylamide gel and electroblotted onto polyvinylidene fluoride membranes. Membranes were incubated in 5% non-fat milk 2 hours and then incubated with primary antibodies, including SOX2, OCT4, Nanog, ABCG2 and β-actin (Zsbio, Beijing, China) at 4°C overnight. After washing with Tris-buffered saline Tween 20 buffer, the membranes were incubated with secondary antibodies conjugated by horseradish peroxidase (HRP) for 2 hours at room temperature. Enhanced chemiluminescence reagent (HaiGene, Harbin, Heilongjiang, China) were used to visualize proteins. (Information of antibodies was provided in Table S1).

The total cellular samples were washed twice with PBS and lysed in RIPA buffer (HaiGene) supplemented with 1 mM PMSF (Sigma-Aldrich, St. Louis, MO). The concentration of total protein was determined using BCA Protein Assay Kit (Pierce, Rockford, IL). The total cellular protein extracts were separated by SDS–PAGE and transferred onto the nitrocellulose membrane (PALL, Washington, NY) in 20 mM Tris–HCl (pH 8.0) containing 150 mM glycine and 20% (v/v) methanol. The membrane was blocked with 5% nonfat dry milk in 1 × TBS containing 0.05% Tween 20 and incubated with primary antibody at 4 °C overnight. Antibodies against Akt (1:1000), phospho-Akt (Ser473) (1:500) were purchased from Cell Signaling Technology. Antibodies against ASK1 (1:500), phospho-ASK1 (Thr845) (1:500), p38 (1:1000), phospho-p38 (Tyr182) (1:500), β-Actin (1:1000) and secondary antibodies (1:5000) were all purchased from Santa Cruz (Weatherford, TX). Occludin antibody (1:1000) was obtained from Invitrogen, and ZO-1 antibody (1:1000) was obtained from Abcam (Cambridge, MA). The signals were developed with enhanced chemiluminescence reagent (HaiGene), and the digital images were captured using a LAS-4000 CCD camera system (Fuji film, Tokyo, Japan). The relative intensity of bands was analyzed by the software of Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD).

Cells were lysed with RIPA buffer (Beyotime, China) containing a Protease Inhibitor Cocktail (Roche, Germany) and then centrifuged at 12,000 × g for 15 min at 4°C. Equal amounts of protein were separated via gel electrophoresis and transferred to a PVDF membrane (Millipore Corp, USA). Membranes were blocked with 5% skimmed milk for 1 h at 37°C and incubated with primary antibodies (Table 1) overnight at 4°C and then with corresponding secondary antibodies for 1 h at 37°C. The blotting signal was visualized using enhanced chemiluminescence reagent (HaiGene, China). Final protein levels were normalized to that of GAPDH.