Rabbit anti β catenin antibody

Whole-cell lysates were generated by scraping cultures of matured cardiomyocytes treated with CHIR99021, N-cadherin antibody, or combination for 1 day and resuspended in RIPA lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, 5 M NaCl, and 1 M Tris pH 7.4) for 10 min at 4 °C and then centrifuged at 11,400g for 20 min at 4 °C for the isolation of the cytosolic fractions. Quantification of protein concentration was determined using Pierce BCA Protein Assay kit (Thermo-scientific). Protein lysate of 60 μg per sample was loaded onto 4–20% Mini-PROTEAN TGX Stain-Free Precast Gels (Bio-Rad), resolved by electrophoresis, and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% skimmed milk in TBS + 1% Tween-20 and probed overnight with primary antibodies. The antibodies were rabbit anti-β-catenin antibody (1:2500; Abcam ab32572) and mouse anti-β-actin antibody (1:1000; Cell Signaling). Membranes were washed and incubated for 1 h with their respective IgG-HRP-conjugated secondary antibody (Santa Cruz) in 5% skimmed milk in TBS + 1% Tween-20 and developed using ECL substrate (Bio-Rad).

Proteins were isolated using a total protein extraction kit from Apply gen Technologies Inc., (Beijing, China). The protein concentration was determined using a BCA Protein Assay reagent kit (Novagen, Madison, WI, USA). Equal amounts of protein (100 μg) were separated by Tris-glycine SDS-PAGE and transferred to polyvinylidene fluoride (PVDF). The membranes were then incubated with blocking buffer (5% nonfat dry milk) at room temperature for 1 h and incubated with the followed primary antibodies overnight at 4 °C: Rabbit polyclonal anti-GAPDH antibody (1:10000,Cell Signaling Technology, Danvers, MA, USA); Rabbit anti-β-catenin antibody (1:5000, Abcam); Mouse anti-GSK-3β antibody (1:1000, Abcam). Then, the membranes were incubated with the corresponding fluorescent labeling secondary antibody (1:8000, Rockland, Gilbertsville, PA, USA) for 1 h at room temperature. The relative density of each band was analyzed on an Odyssey infrared scanner (LICOR Bioscience, Lincoln, NE, USA). GAPDH was used as an internal control.

β-catenin staining was performed at 4 °C overnight using a Abcam rabbit anti-β-catenin antibody (Cambridge, MA). Biotinylated secondary antibody in combination with streptavidin fluorescein (Vector Labs) was used for visualization. Mounting media with DAPI (Vector Labs) was used as a counterstain. All images were taken in Olympus BX-63 microscope with the help of Cell Sens software from Olympus America (Center Valley, PA).