Sc7199

Western blot analysis was carried out as previously described (Telias et al., 2015b (link)). Protein was extracted using reporter lysis buffer (Promega), and 25–30 μg of protein were loaded on a 7.5% separating gel using Mini Trans-Blot Cell (Bio-Rad). Nitrocellulose membranes were stained with primary antibodies against human β-catenin (Santa Cruz, #sc7199, 1:250 dilution) and human β-Actin (Abcam, #ab8224, 1:500 dilution), and detected with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, 1:10,000 dilution). Protein bends were detected using EZ-ECL (BioInd.). Gel images were analyzed using ImageJ (NIH). Identical regions of interest were drawn around β-catenin and β-Actin gel bands across samples and cell lines, and mean gray value was measured in all of them.

Normal cells and cells transfected with plasmids for 24 h were fixed with 4% paraformaldehyde for 15 min and then perforated by 0.2% Triton X-100. Cells were incubated with the first antibodies for 20 h at 4°C and then with Alexa Fluor 594/488-conjugated secondary antibody for 1 h, and DAPI was used as the nuclear counterstain. All the above operations between different reagents were washed three times with PBS. An inverted fluorescence microscope photographed the typical areas. Antibodies used were as follows: anti-β-catenin (Santa Cruz, sc7199, 1:200), anti-CD133 (Abcam, ab276130, 1:200), and anti-ALDH1 (CST, 54135S, 1:200).

Cells and fresh myocardial tissues from the myocardial infarction (MI) area were washed three times with cold PBS and lysed with the RIPA buffer (Beyotime, P0013B) containing protease and phosphatase inhibitors (Boster, AR1183) according to the manufacturer’s instructions. A BCA protein assay kit (Beyotime, P0011) was used to determine the protein concentration. Equal amounts of protein were loaded on the wells of each line in a 10% SDS-PAGE gel for electrophoresis and then transferred to a PVDF membrane. The membranes were then blocked with 5% BSA in TBST buffer for 1 h at room temperature, and incubated with a primary antibody against caspase-3 (CST, 9662s), BAX (CST, 2774), BCL-2 (Abcam,196495), OCT4 (Boster,bs-0830R), p-AKT (CST, 13038S), AKT (CAT, 2920), ERK1/2 (CST, 9102), p-ERK1/2 (CST, 4370S), p-β-catenin (ser33/37/T41, CST, 9561s), β-catenin (Santa, sc7199), SFRP2 (Abcam, ab86329), and NFAT4 (Abcam, ab93628) at 4 °C overnight. Following three washes with TBST, the membranes were incubated with their corresponding secondary antibody (Jackson 111-035-003 and Jackson 111-005-003) at room temperature for 1 h, and the blots were visualized using chemiluminescence (ECL; Forevergen Biosciences Center, Guangzhou, China). Blots were quantified by densitometric scanning. The level of protein expression was normalized against GAPDH controls.