Anti nkx 2.5 sc 8697

Western blotting was performed as described previously52 (link). Briefly, protein lysates were prepared in RIPA buffer supplemented with complete protease inhibitor (Roche Applied Science). Proteins were then separated on 6–15% SDS-PAGE, transferred onto PVDF membranes, and probed with anti-CIBZ [amino acids 1184–1197 (EQKDDIKAFAENVL) of CIBZ]17 (link), anti-Oct3/4 (MAB1759, R&D Systems), anti-Sox2 (S1451, Sigma-Aldrich), anti-Nanog (AB5731, Millipore), anti-α-tubulin (clone DM 1A, Sigma-Aldrich), anti-Brachyury (sc-17743, Santa Cruz Biotechnology), anti-Nkx-2.5 (sc-8697, Santa Cruz Biotechnology), anti-Islet 1 (ab109517, Abcam), anti-cTnI (ab19615, Abcam), and anti-MHC (clone MF20, Developmental Studies Hybridoma Bank) antibodies. HRP-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling) were used as secondary antibodies.

The differentiated cardiomyocytes plated on fibronectin-coated dishes were washed once with PBS, and fixed with 4% Paraformaldehyde Fixative (MUTO Pure Chemicals) at 4°C for 30 min. After fixation, cells were treated with 0.4% Triton X-100 in PBS for 15 min at room temperature. After blocked with ImmunoBlock (DS Pharma Biomedical) for 5 min three times, cells were incubated at 4°C overnight with the primary antibodies, followed by washing with the blocking medium and incubation at room temperature for 60 min with the corresponding secondary antibodies. The immunostaining was performed using the following primary antibodies and reagents: anti-α-Actinin (A7811, Sigma-Aldrich), anti-ANP (sc-20158, Santa Cruz), anti- Troponin-I (ab52862, Abcam), anti-GATA4 (sc-1237, Santa Cruz), anti-Nkx2.5 (sc-8697, Santa Cruz), anti-Titin (HPA007042, Sigma-Aldrich), anti-SERCA2 (MAB2636, Millipore), anti-Connexin43(c6219, Sigma-Aldrich), anti-Na/Ca exchanger(MA3-926) and 4′,6-Diamidino-2-Phenylindole (DAPI, Molecular Probes). The secondary antibodies used were anti-rabbit IgG and anti-mouse IgG or IgM conjugated with Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes). Signal was detected using a conventional fluorescence laser microscope equipped with a colour charge-coupled device camera (BZ-9000, KEYENCE).