Anti nanog

Western blot was performed as previously described 44 (link). The antibodies included anti-Oct4 (Bioss, Beijing, China, bs-0830R, 1:1000), anti-Nanog (Proteintech, Wuhan, China, 14295-1-AP, 1:1000), anti-CD44 (Proteintech, 15675-1-AP, 1:1000), anti-Sox2 (Proteintech, 11064-1-AP, 1:1000), anti-γ-H2AX (Abcam, Cambridge, UK, ab26350, 1:1000), anti-DLG2 (Affinity Biosciences, Suzhou, China, DF3995, 1:1000), anti-p-Yap1 (CST, Boston MA, USA, 4911S, 1:1000), anti-Yap1 (Proteintech, 13584-1-AP, 1:1000), anti-Bax (CST, B8429, 1:1000), anti-Bcl2 (Proteintech, 60178-1-Ig, 1:1000), anti-β-tubulin (Sigma-Aldrich, T5201, 1:1000), anti-Lamin B1 (Santa, Dallas TX, USA, sc-365962, 1:1000), anti-TEAD1 (ABclonal, Wuhan, China, A13366, 1:1000), anti-β-actin (Santa, sc-8432, 1:4000), CD63 (Proteintech, 25682-1-AP, 1:1000), and TSG101 (Proteintech, 28283-1-AP, 1:1000).

Proteins were resolved using 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of separated proteins onto a nitrocellulose membrane. Non-specific antigens on the membrane were blocked by incubating the membrane in 1×TBST (Tris-buffered saline with 0.1% Tween-20) containing 5% non-fat skim milk at room temperature for 1 h. Afterward, the membrane was incubated overnight with primary antibodies at 4°C followed by incubation with the secondary antibody (goat anti-mouse or anti-rabbit IgG antibody) at room temperature for 1 h. The primary antibodies used was anti-caspase3 (Abcam, US, 1;500), anti-Bax (R&D system, US, 1:1000), anti-Bcl2 (Abcam, US, 1;500), anti-Sox2 (Abcam, US, 1:1000), anti-Sox9 (Abcam, US, 1:1000), anti-CD133 (Proteintech, US, 1;1000), anti-Nanog (Proteintech, US, 1:1000), anti-SOCS2 (Abcam, US, 1:1000), anti-SOCS5 (Abcam, US, 1:500), anti-PTPN1 (Proteintech, US, 1:2000), anti-PTPN11 (Proteintech, US, 1:500), anti-STAT3 (Proteintech, US, 1:1000) The immunoblots were developed using the BeyoECL kit (Beyotime, China) and Tanon 5200 system (Tanon, China).

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.