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Tissues were fixed in 4% paraformaldehyde (PFA) for 0.5 h, permeabilized with 0.5% (v/v) Triton X-100 in Dulbecco’s PBS (D-PBS) for 1 h, and immersed in blocking solution at 4°C overnight. The tissues were then incubated with the primary antibodies anti-α-actinin (1:1000; A7811; Sigma-Aldrich), anti-troponin T2 (TnT2; 1:200; SC-20025; Santa Cruz Biotechnology, Dallas, TX, USA), anti-connexin 43 (Cx43; 1:200; C6219; Sigma-Aldrich), or anti-β-MHC (1:100; SC-53089; Santa Cruz Biotechnology) at 4°C overnight. Thereafter, the tissues were rinsed with PBS and incubated with the secondary antibodies Alexa Fluor 594 anti-mouse IgG (715-586-150; Jackson Immuno Research, West Grove, PA, USA), DyLight-594 anti-mouse IgM (715-516-020; Jackson Immuno Research), Alexa Fluor 647 anti-rabbit IgG (A21245; ThermoFisher), and Alexa Fluor 488 anti-rabbit IgG (A21206; ThermoFisher) at a dilution of 1:300 in blocking buffer at room temperature for 1 h. DAPI (300 nM; Wako Pure Chemical Industries, Ltd.) was used to stain the nuclei for 30 min. Images were captured using a confocal microscope (NIKON A1; Nikon).

Whole protein from NRCMs or mouse heart tissue was extracted as previously reported 11 (link). Intercellular signaling was analyzed using a Pathscan Intercellular Signaling Array kit (#7323 and #12856; Cell Signaling Technology, Inc.) following the protocol provided, and routine western blotting procedures were performed according to our previous report 11 (link). The primary antibodies used were as follows: anti-ANP (mouse mAb; 1:300; sc-515701; Santa Cruz Biotechnology, Inc.), anti-BNP (rabbit pAb; 1:500; ab19645; Abcam), anti-MYH7 (mouse mAb; 1:300; sc-53089; Santa Cruz Biotechnology, Inc.), anti-Erk1/2 (rabbit mAb; 1:1000; #9102), anti-phospho-Erk1/2 (Thr202/Thr204) (rabbit mAb; 1:1000; #9102), anti-Akt (rabbit mAb; 1:1000; #9272), anti-phospho-Akt (Ser473) (rabbit mAb; 1:1000; #4060), anti-PKC (rabbit mAb; 1:1000; #2056), anti-phospho-PKC pan (Thr514) (rabbit mAb; 1:1000; #9379), anti-AMPK (rabbit mAb; 1:1000; #5831), anti-phospho-AMPK (Thr172) (rabbit mAb; 1:1000; #2535), and anti-GAPDH (rabbit mAb; 1:2000; #2118;Cell Signaling Technology, Inc.). The secondary antibodies used were horseradish peroxidase (HRP) conjugated (GE Healthcare Life Sciences, Beijing, China): anti-mouse IgG, HRP-conjugated whole Ab sheep (NA931) or anti-rabbit IgG, HRP-linked whole Ab donkey (NA934).