Ab115262

The differentiated hiPSCs on D20 were plated on 12-well culture dishes. The cells were harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of proteins were loaded onto a gel for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred onto a nitrocellulose membrane, and incubated with primary antibodies against HCN4 (ab32675; Abcam, Cambridge, MA, USA), TBX18(ab115262; Abcam, Cambridge, MA, USA), ISL-1(abs132916; Abcam, Cambridge, MA, USA) TBX3(ab154828; Abcam, Cambridge, MA, USA), TBX5(Gene-Tex; Cat No. GTX113849), α-tubulin (GB11200; Service-bio, Wuhan, China), GAPDH antibody (Abcam; Cat. no. ab37168) and Shox2(ab55740; Abcam, Cambridge, MA, USA) overnight at 4 °C. The primary antibodies were detected by incubating the membrane with secondary antibodies for 1 h, and then enhanced chemiluminescence detection (ECL; Beyotime Institute of Biotechnology) was performed. The level of α-tubulin or GAPDH was considered to normalize the signal intensities. The Western blot assay was repeated at least three times to verify the results.