Anti anp

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Big Cabin, OK, USA). Collagenase and trypsin were purchased from Sigma (St. Louis, MO, USA). Iso and metformin were purchased from Sigma. The following antibodies were used in this study: anti-Mfn2, anti-PINK1 (Abcam, Cambridge, UK), anti-Beclin1, anti-P62, anti-LC3B, anti-PGC 1α, anti-TFAM, anti-NRF1, anti-ANP, anti-β-MHC, anti-Col-1, anti-TGF-β-1, anti-GAPDH and anti-β-actin (Proteintech, Chicago, IL, USA).

Proteins were extracted, quantified and analyzed as reported previously (Xiong et al. 2018 (link); Liu et al. 2019 (link); An et al. 2021 (link)). The primary and secondary antibodies used included anti-β-MHC, anti-ANP, anti-HO1, anti-GPX4 (1:1000, Proteintech, USA), anti-PINK1 (1:200, Santa Cruz, USA), anti-SIRT1 (1:1000, Cell Signaling Technology, USA), goat anti-rabbit and anti-mouse IgG-HRP (1:5000, Fudebio, China) antibodies.

The samples were lysed using a mixture of radioimmunoprecipitation assay lysis buffer, phosphatase inhibitor, and phenylmethylsulfonyl fluoride (100:1:1) and an Ultrasonic Cell Disruptor. The lysates were subjected to electrophoresis. The resolved proteins were transferred to a polyvinyl difluoride membrane (Millipore, USA). The membrane was blocked with 5% milk powder in Tris-buffered saline containing Tween-20 (TBST) for 1 h. Next, the membrane was probed with the anti-ANP (1:1,000, Proteintech, China) antibodies at 4°C overnight, followed by incubation with the horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h. After washing the membrane thrice with TBST, immunoreactive signals were developed using an enhanced chemiluminescence reagent (Fude Biological, Hangzhou, China).