Anti atg7

TSA was purchased from Enzo Life Sciences. Carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC) and 7-amino-4-trifluoromethyl coumarin (AFC) were from Enzyme Systems Products (Livermore, CA). Unless indicated, all other reagents including cisplatin and chloroquine were purchased from Sigma (St. Louis, MO). The following primary antibodies were used: anti-LC3B from Novus Biologicals (Littleton, CO); anti-ATG7, anti-β-actin and anti-cyclophilin B from Abcam; and anti-cleaved caspase3, anti-AMPK, anti-phospho-AMPK (Thr172), anti-P70S6K, anti-phospho-P70S6K (T389) from Cell Signaling Technology (Danvers, MA). All secondary antibodies for immunoblot analysis were from Thermo Scientific (Rockford, IL).

Reagents were purchased as follows: Cabergoline (Catalog #2664, Tocris, Bristol, UK); anti-phospho-p70s6k (Catalogue #9208), anti-p70s6k (Catalogue #2708), anti-phospho-4EBP1 (Catalogue #2855), anti-4EBP1 (Catalogue #9644), anti-phospho-mTOR (Catalogue #5536), anti-mTOR (Catalogue #2983) and PARP (Catalogue #9542) were from Cell Signaling Technology (Danvers, MA); Anti-β-tubulin (Catalogue #ab151318), Anti-GAPDH (Catalogue #ab181602), anti-ATG7 (Catalogue #ab133528), and anti-p62 (Catalogue #ab56416) were from Abcam (Cambridge, MA); LC3 (Catalogue #L7543) was from Sigma-Aldrich (St. Louis, MO); Becn1 (Catalogue #PD017) was from MBL (Woburn, MA). All antibodies were used according to manufacturers' instructions.

For total protein extraction, samples were ground in liquid nitrogen and homogenized in ice-cold extraction buffer (50 mM sodium phosphate (pH 7.0), 200 mM NaCl, 10 mM MgCl₂, 0.2% (v/v) β-mercaptoethanol, 10% (v/v) glycerol) with protease inhibitor cocktail (Roche). To collect the supernatant for electrophoresis, extracts were incubated on ice for 30 min and centrifuged at 12,000× g for 30 min at 4 °C. For immunoblot analysis, total proteins were subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose Western blotting membranes (Amersham) with a pore size of 0.2 µm or 0.45 µm PVDF (Immobilon, Merck, Germany). Immunoblot analysis was performed using anti-ADH1 (catalog no. AS10685, Agrisera, Sweden), anti-PDC1 (catalog no. AS10691, Agrisera, Sweden), anti-GFP (catalog no. 2955, CST, USA), anti-ATG7 (catalog no. ab53255, Abcam, UK), anti-ATG8a (catalog no. ab77003, Abcam, UK), and anti-ACTIN (catalog no. 58169, CST, USA) as the primary antibodies and horseradish peroxidase-conjugated anti-rabbit lgG (catalog no. A21020, Abbkine, China) and anti-mouse lgG (catalog no. A21010, Abbkine, China) as the secondary antibodies.

Western blot analysis was performed as previously described [36 (link)]. Total protein was extracted from the H9c2 cells and rat myocardium using radioimmunoprecipitation assay lysis buffer containing protease and phosphatase inhibitors (Beyotime, Beijing, China) and quantified using a BCA protein assay. Approximately 30 g of protein was loaded and separated on an 8% SDS-PAGE gel, and then transferred to polyvinylidene difluoride membranes (BIO-RAD, Hercules, USA). The membranes were blocked with nonfat milk for 2 h at room temperature, and then incubated with primary antibodies at 4 °C overnight. Subsequently, the membranes were washed in PBS with Tween-20 before incubating with secondary antibodies for 2 h at room temperature. The antigen–antibody bands were visualized and quantified using ImageJ software (Bethesda, MA, USA). The primary antibodies used in this study and corresponding dilution ratios were as follows: anti-alpha Tubulin (1:1000), anti-Atg7 (1:500), anti-LC3 (1:1000), anti-P62 (1:1000), anti-HIF-1α (1:1000) (Abcam, Cambridge, USA).

Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 was purchased from Sigma (catalog no. L2630, St. Louis, MO). The sources of the primary antibodies were as follows: anti-LC3 from Novus Biologicals (Littleton, CO), anti-Atg7, anti-Atg5 and anti-p62 from Abcam (Cambridge, MA), anti- GAPDH from Sigma (St. Louis, MO). All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories Inc (West Grove, PA). Other reagents, including choloquine and paraformaldehyde, were from Sigma (St. Louis, MO).

Protein levels were analyzed using IB and ELISA following conventional protocols. For IB, the primary antibodies used were anti-YAP (Abcam, Cambridge, MA, USA, #ab52771), anti-GAPDH (CST, Boston, MA, USA, #5174 and #51332), anti-p-127-YAP (Abcam, #ab76252), anti-p-381-YAP (CST, #13619), anti-O-Glc-YAP (developed by Biolynx, Hangzhou, China), anti-HRS (Abcam, #ab72053), anti-PLD2 (CST, #13904), anti-RAB2B (Novus, Littleton, CO, USA, #NBP1-31631), anti-RAB27A (Abcam, #ab55667), anti-RAB27B (Abcam, #ab103418), anti-VAMP (Abcam, #ab36195), anti-ATG7 (Abcam, #ab52472), anti-SOX10 (Abcam, #ab227680), anti-Mycn (Abcam, #ab227822), anti-ALYREF (Abcam, #ab202894), anti-NSUN2 (Abcam, #ab259941), anti-DNMT2 (Abcam, #ab220175), anti-NSUN1 (Abcam, #ab270175), anti-NSUN3 (Abcam, #ab272616), anti-NSUN4 (Abcam, #ab235430), anti-NSUN5 (Abcam, #ab121633), anti-NSUN6 (Novus, #NBP1-92202), anti-NSUN7 (Biorbyt, Cambridge, UK, #orb258175), anti-YBX1 (Abcam, #ab255606), anti-EXOSC10 (Abcam, #ab94981), anti-SPI1 (Abcam, #ab227835), anti-CD63 (Abcam, #ab271286), anti-TSG101 (Abcam, #12501), anti-ALIX (Abcam, #ab275377), anti-CD9 (Abcam, #ab92726), and anti-calnexin (Abcam, #ab133615). For ELISA, YAP and NSUN2 levels were measured using kits from Yingxin Biotech Ltd. (Shanghai, China) as per manufacturer’s instructions.

Thapsigargin and BFA were purchased from Sigma-Aldrich (St Louis, MO, USA). Apoptozole was purchased from Millipore (Billerica, MA, USA). Anti-pendrin (G-19, epitope against C terminus), anti-HA, anti-Myc, anti-aldolase and anti-Flag antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-pendrin (ab66702, epitope against N terminus), anti-calnexin, anti-giantin, anti-Lamp1, anti-CHMP2B, anti-ATG7 and anti-Hsc70 were from Abcam (Cambridge, UK); anti-EEA1 was from BD Biosciences; anti-CD9 was from LSBio (Seattle, WA, USA); anti-DNAJC14 was from Novus (Littleton, CO, USA); anti-Rab7 was from Cell Signaling Technologies (Danvers, MA, USA); and anti-CFTR antibody (M3A7) was from Millipore. Detailed information for primary and secondary antibodies used in this study is summarized in Supplementary Table 4.