Atp6v1a

Cells were lysed with RIPA buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 1% Triton X‐100, 0.25% deoxycholate, and 0.1% SDS) supplemented with protease inhibitor (Complete, Roche). The 20 μg of protein per lane were separated by SDS‐PAGE and subsequently transferred to nitrocellulose membrane. After semidry blotting membranes were blocked 1 hour with blocking buffer (LI‐COR Biosciences) and probed with an antibody against ATP6V1A (1:1000, Abcam) and GAPDH (1:40 000, Ambion) overnight. Membranes were washed and incubated with HRP‐ or IRDye‐conjugated secondary antibodies. Signals were detected with Odyssey FC Imaging System and densitometric quantification was performed using Image Studio (LI‐COR Biosciences). Significance levels were calculated using Student's t‐test.

The antibodies used in our experiments included: ATP6V1A (Abcam, ab199326), EGF receptor (Cell Signaling Technology, 4267), LC3 (microtubule-associated protein 1 light chain 3) (Sigma, L7543), α-tubulin (Sigma, T6199), Lamin AC (Cell Signaling Technology, 2032), LAMP1 (Cell Signaling Technology, 9091), SQSTM1 (Sigma, SAB1406748), TFEB (Bethyl Laboratories, A303-673A).
The chemicals used in our experiments were: Annexin V Pacific Blue™ conjugate (Thermo Fisher Scientific, A35122), acridine orange (AO) (Immunochemistry Technologies, LLC, 6130), docetaxel (Sigma, 01885), chloroquine (CQ) (PubChem, 2719), lysoTracker Red DND-99 (Invitrogen, L7528), lysoSensor green DND-189 (Invitrogen, L7535), Magic Red^TM cathepsin B and L reagent with Acridine Orange (Immunochemistry Technologies, LLC, 937/938/6130), thapsigargin (Sigma, T9033).

The myocardial tissues were lysed with lysis buffer containing using protease and phosphatase inhibitors. Total protein content was assessed using BCA reagent. Equivalent amounts of protein extract were separated on SDS-PAGE, and subsequently transferred onto PVDF membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies such as ANP (Abcam, ab180649), GPX4 (Abcam, ab125066), NOX1 (Abcam, ab131088), ACSL4 (Abcam, ab155282), FTH1 (Abcam, ab183781), ATP6V1A (Abcam, ab199326), and GAPDH (Cell Signaling, 2118) overnight at 4°C. After washing, the membranes were incubated with HRP-conjugated secondary antibodies and visualized using an enhanced chemiluminescence substrate kit (Bio-Rad Laboratories).