Ab84593

Immunohistochemical staining for GATA4 expression was performed as previously described (13 (link),14 (link)), using a primary antibody against GATA4 (ab84593; 1:100; Abcam). The endothelial layer of the thoracic aorta was sliced into 6–8 µm thick sections. Primary antibodies were detected using a HRP conjugated goat anti-rabbit secondary antibody was from (ab6721; 1:2,000; Abcam). Positive expression was assessed by determining the % of positive GATA4 staining in the entire visual field.

Endothelin receptor type A (ETRA) antagonist BQ123 and endothelin receptor type B (ETRB) antagonist BQ788 were purchased from Sigma (St. Louis, MO, USA). ET-1 (human, porcine) was purchased from Tocris Bioscience (Minneapolis, MN, USA). The NOX4 inhibitor GLX351322, secreted phospholipase (sPLA2) inhibitor varespladib, cytosolic PLA2α (cPLA2α) inhibitor CAY10650, ROS inhibitor N-Acetyl-D-cysteine (NAC), Src antagonist Src inhibitor 1, extracellular signal-regulated kinase (ERK) antagonist PD98059, and protein kinase B (Akt) antagonist LY294002 were purchased from Med Chem Express (Monmouth Junction, NJ, USA). Anti-NOX4 and anti-GATA4 antibodies were obtained from Abcam (ab133303, ab85163, ab117529, and ab84593, respectively; Cambridge, UK). The anti-Src antibody was from Millipore (04-889; Burlington, MA, USA). Anti-NOX2 antibody was obtained from Bioss (bs-3889R; Beijing, China). Anti-Akt, phospho-Akt (S473), and β-actin antibodies were acquired from Bioworld Technology (BS1008, BS4006, and AP0060, respectively; Nan Jing, China). Anti-ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), cPLA2, and phosho-cPLA2 (Ser505) antibodies were obtained from Affinity (AF0155, AF1015, AF6329, and AF3329, respectively; Chang Zhou, China).

Cell lysates were collected from undifferentiated D3G4 mESC lines in RIPA lysis buffer supplemented with protease inhibitors (Roche). Equal amounts of protein were loaded on a 10% polyacrylamide gel and after electrophoresis, were transferred by semi-dry transfer onto a PVDF membrane (Millipore). The membrane was washed in Tris-buffered saline (TBS) (20 mM Tris, 0.5 M NaCl, pH 7.5), blocked for 1 h at room temperature in TBS-T (0.5% [vol/vol] Tween 20 in TBS) supplemented with 10% (vol/vol) nonfat dry milk, and incubated overnight at 4°C with a 1:100 dilution of anti-GATA4 antibody ab84593 (Abcam) in blocking buffer. The following day, the membrane was washed 3 times for 10 min each in TBS-T, incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-mouse secondary antibody (RABHRP1, Sigma) diluted in blocking buffer for 1 h at room temperature, and developed using SuperSignal^™ West Dura Chemiluminescent Substrate (Thermo Scientific).

EBs were digested into single cells by incubating with 0.25% trypsin (Invitrogen) plus 0.25% collagenase II (Invitrogen, 1:1) for 30 min at 37°C. The isolated single cells (1 × 10⁶) were then immunostained with the antibodies listed below in PBS with 0.2% Tween 20. After cell aggregates were removed, single cells were analyzed on the BS LSRII flow cytometer (BD Bioscience) and analyzed using CellQuest software (BD Biosciences). Polyclonal rabbit IgG isotype control (Abcam, ab171870) was included in all experiments. The following antibodies were used: anti-Tbx5 (Abcam, ab101227), anti-GATA4 (Abcam, ab84593), and Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (Invitrogen, A-21206).