Sc 10768

Immunohistochemical staining was carried out using a rabbit anti-BRG1 polyclonal antibody (sc-10768, Santa Cruz, USA, diluted 1:250) and a rabbit anti-WNT3A polyclonal antibody (ab19925, Abcam, Cambridge, United Kingdom, diluted 1:100). Positive staining was scored by two independent investigators who had no knowledge of the patient outcomes. When discrepancy in an assessment was encountered, the slides were re-examined by both pathologists under a multi-head microscope to obtain agreements. The staining intensity was scored as being 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The extent of staining was scored according to the percentage of positively stained cells: 0 (0–10%), 1 (11–25%), 2 (26–75%), and 3 (75–100%). The sum of the intensity and extent of the scores were defined as the final staining score. The specimens were divided into 2 groups according to their overall scores, which were as follows: 0–3 = negative and weak, 4–6 = moderate and strong positive.

Total proteins were extracted from cultured cells and tissues using RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China). Protein concentrations were determined using BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China). Equal amounts of protein were separated by electrophoresis on SDS polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk solution for 1 h, and then incubated with a primary antibody overnight at 4°C and a secondary antibody at room temperature, successively. The bands were detected by ECL chemiluminescence (Millipore, USA) according to the manufacturer’s instructions. Expression of GAPDH or actin was applied as internal control to confirm equal loading of whole protein.
Antibodies used in the Western blot assay were as follows: anti-BRG1 polyclonal antibody (sc-10768, Santa Cruz, USA, diluted 1:250), anti-WNT3A polyclonal antibody (ab19925, Abcam, Cambridge, United Kingdom, diluted 1:100), anti-GAPDH antibody (sc-137179, Santa Cruz; USA, diluted 1:2000), anti-actin antibody (60008-1-Ig, Proteintech; USA, diluted 1:2000).

For co-immunoprecipitation cardiac progenitor cells differentiated from mouse ES cells and 30 mouse embryos (E8.25-E9) homogenized with plastic pestle were lysed with lysis buffer (50 mM Tris pH 7.5, 10 mM EGTA, 100 mM NaCl, 0.1% Triton X-100) plus protease and phosphatase inhibitors on ice. After sonication and centrifugation, the supernatant was used for immunoprecipitation. Embryoid bodies were trypsinized to obtain a single cell suspension. Nuclei were isolated by incubating in lysis buffer containing 50 mM Tris pH 8.0, 2 mM EDTA pH 8.0, 0.1% NP-40, 10% glycerol along with protease and phosphatase inhibitors. Isolated nuclei were disrupted by sonication in 50 mM Tris pH 8.0, 0.1% SDS, 5 mM EDTA pH 8.0 buffer along with protease and phosphatase inhibitors. 500 µg of protein lysates were precleared with Protein G-Sepharose beads (GE Healthcare, 17-0618) at 4 °C on a rotary shaker for 1 h. Precleared lysates were incubated with 1 µg of α-Isl1 antibody (39.4D5 DSHB) overnight at 4 °C. Protein complexes were bound to Protein G-Sepharose beads for 1 h at 4°C. Protein complex bound beads were washed and resolved in a standard SDS-PAGE system. Brg1 (1:500, Santa Cruz, SC-10768), Isl1 (1:10 dilution; 39.4D5 DSHB) and Baf60c antibody (1 to 500 dilution; a kind gift from Pier Lorenzo Puri) were used for western blotting.