Ab154841

Cells and tissues were lysed in RIPA buffer. Histones were extracted as described below. Samples were subjected to western blot analysis as reported [20 (link),117 (link)]. Primary antibodies were: α-tubulin (T6074, Sigma-Aldrich), SMAD3 (C67H9, Cell Signaling, Danvers, MA, USA), pSMAD3 (C25A9, Cell Signaling), α-SMA (F3777, Sigma-Aldrich), VASP (3112, Cell Signaling), pVASP (Ser239) (3114, Cell Signaling), GUCY1B3, sGC1β (ab15484) (ab154841, Abcam), PKG-1 (3248, Cell Signaling), histone-3 (05-928, Millipore), Acetyl-Histone H3 (Lys14) (06-911, Millipore), pAKT (Ser473) (9271, Cell Signaling), AKT (9272, Cell Signaling), active β-catenin (05-665, Millipore), collagen-I (ab138492, Abcam, USA), and DKK1 (AF1096, R&D Systems). For quantification of immunoblot signals we used the Image Lab Software from Bio-Rad Laboratories.

Protein was extracted from isolated neonatal cardiomyocytes using RIPA buffer (Sigma-Aldrich, Milwaukee, WI, USA) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche, Rotkreuz, Switzerland). Total protein quantification was determined using BCA protein assay (Sigma-Aldrich, Milwaukee, WI, USA). 40µg of total control or MDX protein were loaded into NuPAGE 4–12% Bis-Tris Gel (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). The membrane was then incubated with antibody against the β1-subunit of sGC (GUCY1B3), from Abcam (ab154841) at a 1:250 dilution and the GAPDH antibody from Proteintech (60004-1-Ig) at 1:1000 dilution.

Lung tissues were weighted and lysed in ice-cold RIPA buffer (P0013B, Beyotime) supplemented with phosphatase inhibitor complex (C500017-0001, Sangon) and protease inhibitor cocktail (C600387-0001, Sangon) according to the manufacturer’s instruction. After homogenization, samples were slowly rotated at 4 °C for 15 min for complete lysis. Then samples were centrifuged and the supernatants were collected. Protein concentrations were determined using the BCA assay (20201ES86, Yeasen). Proteins were separated using SDS polyacrylamide gels and transferred onto nitrocellulose membranes (10600001, Amersham Protran). The membranes were blocked with 5% BSA (A600332, Sangon) in TBS-T buffer (containing 0.1% Tween-20) for 1 h. Then incubated with primary antibodies at 4 °C overnight, and secondary antibody at room temperature for 1 h. The following primary antibodies were used in this study: Rabbit anti-β-ACTIN antibody (1:1000, 4967, Cell Signaling Technology), Rabbit anti-GUCY1A1 antibody (1:500, G4280, Sigma), Rabbit anti-NOTCH3 antibody (1:500, ab23426, Abcam), Rabbit anti-GUCY1B1 antibody (1:1000, ab154841, Abcam). HRP-conjugated donkey anti-rabbit IgG (H + L) (1:10000, 711-066-152, Jackson ImmunoResearch) was used as secondary antibodies. Western blot was performed using Pierce™ ECL Western blotting substrate (32209, Thermo) according to the manufacturer’s instruction.