Anti ctnt

Total protein samples were prepared in radioimmunoprecipitation assay (RIPA) buffer; then, the protein concentration was determined by BCA assay (Beyotime). The total protein were separated by electrophoresis through Future PAGETM 4–12% (ACE, 11 Wells), then transferred the protein to PVDF membranes (Millipore, 0.45 µm), blocked with 8% skim milk and incubated with anti-PYGO2 (1:1000, Genetex), anti-NXK2.5 (1:1000; Invitrogen), anti-GATA4 (1:1000, Proteintech), anti-cTnT (1:1000, Abcam), anti-β-catenin (1:1000, Proteintech), anti-β-Tubulin (1:5000, ABcanol), anti-ACTA2 (1:1000, Abcam), anti-GAPDH (1:5000, ABcanol), and anti-β-ACTIN antibody (1:5000 dilution, ABcanol). The signal densities of the target protein bands were quantified and normalized to β-ACTIN or GAPDH using Image J. Nucleo-cytoplasmic isolation was performed according to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech), followed by routine protein isolation and antibody incubation. As for the analysis of nuclear localization of β-catenin, we used the formula [(β-catenin/H3)-(Protein/GAPDH) + 1] (in both the vector and PYGO2 groups) to exclude for cytopasmic contamination.

Following 3 washes with PBS, cells (plated on a 1 × 1 cm glass slide) were fixed for 20 min with 4% paraformaldehyde and washed again with PBS. The samples were then incubated for 10 min at room temperature with 0.5% Triton X-100 in PBS and blocked with 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Primary antibodies were diluted in 5% BSA in PBS and incubated overnight at 4 °C. Following 3 washes with 0.5% Triton X-100 in PBS, the samples were incubated with secondary antibodies diluted in 5% BSA for 60 min at 37 °C. The samples were then washed, incubated with Hoechst 33342 (Beyotime, China) in PBS for 30 min at 37 °C and washed again before imaging. Immunofluorescence staining was imaged using an A1R confocal microscope (Nikon, Japan). The following antibodies were used in this study: anti-SOX2, anti-Nanog (Proteintech, China), anti-cTnT, anti-CX43 (Abcam, China), and anti-α-actinin (Proteintech, China). The secondary antibodies were goat anti-rabbit Cy3, rabbit anti-mouse Cy3 (CWBIO, Beijing, China), and goat anti-rabbit 488 (ZSGB-Bio, Beijing, China). Sarcomere lengths and nuclear numbers were measured by ImageJ software. n > 10 cells per condition, three biological replicates. The lengths of ten sarcomeres from each cell were measured and averaged for each condition.

For cardiomyocyte characterization, immunostaining was performed using anti-cTNT (Abcam; 1:100) and anti-alpha-actinin (Sigma-Aldrich; 1:200) as primary antibodies and Alexafluor-488-cojugated anti-mouse IgG as a secondary antibody. Briefly, cells were washed with PBS (Life technologies), fixed with 4 % PFA (Sigma-Aldrich) for 15 min and permeabilized in PBS containing 0.1 % Triton X-100 (Sigma-Aldrich) and 5 % FCS (Life techologies) for 30 min. Cells were then incubated overnight with the primary antibodies. Next day, secondary antibody Alexafluor-488-cojugated anti-mouse IgG (1:1,000; Life technologies) were used to detect and visualize the primary antibodies. All antibodies were diluted in blocking solution. Similar protocol was also used for the staining using ISL1 (Biorbyt; 1:200) antibody. Micrographs were taken with an Axiovert 200 M microscope (Carl Zeiss).

Cells were lysed in lysis buffer (Thermo Fisher Scientific), and protein levels were measured using the BCA method (Thermo Fisher Scientific). Fifty micrograms of proteins was separated by 10–15% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (PVDF, Thermo Fisher Scientific) using a semi-dry blotter. The membrane was blocked with 5% BSA for 1 h and then incubated with specific primary antibodies: anti-TGF-β1, anti-α-SMA, anti-cTnT, anti-p-Smad2/3, anti-Smad2/3, or anti-actin (antibodies were all purchased from Abcam, MA, USA). Following overnight incubation, the membranes were washed with 1% TBST three times (15 min each) and incubated in horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (Abcam) at room temperature for 50 min. Following incubation, the membranes were washed with 1% TBST three times (15 min each). The protein-antibody complexes were detected using the chemiluminescent reagent WesternBright ECL detection kit (Thermo Fisher Scientific). Relative protein levels were determined in comparison with β-actin.

Control and DOX-exposed hiPCS-CMs on coverslips were washed firstly by maintenance medium followed by pre-warm PBS and fixed with 4% paraformaldehyde (10 min, 25 °C). Next, there were rinsed with phosphate saline buffer with 1% Triton X (PBS-T), blocked in 5% BSA for 1 h at 25 °C and incubated with anti-cTnT (Abcam, 1:200) and anti-cTnI (Abcam, 1:200) at 4 °C for one hour. The cells were washed 3 times with cold PBS (each 5 min, 25 °C) and secondary antibodies (Alexa Fluor-488/568-conjugated - Invitrogen-Molecular Probes, Inc., Carlsbad, CA, USA) were added for another 1 h incubation at 4 °C. The cells were again washed 3 times in PBS (each 5 min, 25 °C) and then mounted into Prolong^® Gold anti-fade mount with DAPI (Invitrogen-Molecular Probes, Inc., Carlsbad, CA, USA). Images were taken with a fluorescence microscope Nikon Eclipse E 400 (Nikon, Prague, Czech Republic) and NIS Elements AR software (Nikon, Prague, Czech Republic).