Antibodies against gapdh

Cells were lysed using RIPA buffer (Heart, Xian, China). Cell lysates containing 30 μg of total protein were then subjected to SDS–PAGE (Beyotime, Shanghai, China) and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C (anti-Zic2, Axin2, APC, active-β-Catenin(ser45), β-Catenin, Flag, and c-Myc, 1:1000 dilution; Cyclin D1 and GAPDH, 1:5000). The membrane was then washed six times with TBST buffer for 5 min each and incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) was added to visualize the protein bands. The antibodies against GAPDH were purchased from Santa Cruz (Dallas, TX, USA), the antibodies against Zic2, Cyclin D1, CD44, Axin2, and APC were purchased from Abcam (Cambridge, MA). The antibodies against active-β-Catenin(ser45), β-Catenin, and GSK-3β were purchased from Cell Signaling Technology (Danvers, MA, USA), and the antibodies against Flag was purchased from Proteintech (Rosemont, USA). Nuclear extract was prepared with the protocol in the Nuclear Extraction Kit (Abcam, Cambridge, MA, USA).

NaHS was purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against GAPDH, VEGF, HO-1, and CSE were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against collagen type I and type III, MMP-9 were purchased from Calbiochem (Dermstadt, Germany); antibodies against CD34 and α-SMA were from Abcam (Shanghai, China).

Alkaline phosphatase (AP) staining was performed with the Alkaline Phosphatase Detection Kit (Millipore), following the supplier’s instructions. Immunofluorescence (IF) staining was performed using primary antibodies (all at 1:200 dilutions) to detect Oct4 (Abcam), Sox2 (Abcam), and Nanog (Abcam). Nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI).
Western blot analysis was performed using whole-cell extracts prepared by disrupting the cells in NP40 lysis buffer. Antibodies against GAPDH (Santa Cruz) were used to assess the purity of the respective fractions. The protein bands were visualized using an enhanced chemiluminescence reagent after hybridization with a horseradish peroxidase (HRP)-conjugated secondary antibody. All the Western blot results were quantified by using ImageJ. The antibodies used in this study included anti-Sox2 (Santa Cruz), anti-Sirt1 (Santa Cruz), anti-Myc (Santa Cruz), anti-Oct4 (Santa Cruz), anti-mSSEA1 (Sigma), and anti-Nanog (Cayman).