ATG7 antibody [N3C2], Internal

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	Incubate primary Ab overnight at 4 °C

Protocol tips
Incubate primary Ab overnight at 4 °C

Publication protocol

After treatments, RAW264.7 macrophages were rinsed 3 times with ice-cold PBS and scraped in an ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.5, 0.1% (w/v) Triton X-100 (X100; Sigma-Aldrich) 1 mmol/l EDTA, 1 mmol/l EGTA, 50 mmol/l NaF, 10 mmol/l sodium β-glycerophosphate, 5 mmol/l sodium pyrophosphate, 1 mmol/l sodium vanadate, 0.1% (v/v) 2-mercaptoethanol, and inhibitors (a 1:1000 dilution of protease inhibitor cocktail; P8340; Sigma-Aldrich). The lysates were shaken for 20 min at 4 °C, centrifuged (12,000 × g, 20 min at 4 °C), and the supernatant fraction was collected. Protein concentration was determined using the BCA protein assay (23,227; Pierce, Thermo Fisher Scientific). Proteins were separated on a 10% SDS polyacrylamide gel (ATG7, LAMP-1 and ATP6V0C) or 12% SDS polyacrylamide gel (ATG12, LC3, CTSB and ACTB). Proteins were transferred to nitrocellulose membranes. After non-specific blocking with skim milk for 1.5 h, the membranes were incubated with anti-ATG7 (GTX113613; GeneTex, Irvine, CA, USA), anti-ATG12 (NBP2-43781; Novus Biologicals, Littleton, CO, USA), anti-LC3 (NB100-2220; Novus Biologicals), anti-LAMP-1 (BS-1970R; Bioss Antibodies, Woburn, MA, USA) anti-CTSB (sc-13,985; Santa Cruz Biotechnology, Dallas, TX, USA) or anti-ATP6V0C (sc-134,887; Santa Cruz Biotechnology) overnight at 4 °C. The membranes were then washed three times with Tris-buffered saline added with 0.1% Tween 20 (TBST), and then incubated with an appropriate HRP-conjugated secondary antibody. Membranes were washed three times with TBST, incubated with an ECL solution (Pierce), and exposed to X-ray films. Bands were quantified using GelQuant.NET software and normalized to those of ACTB (A5441; Sigma-Aldrich).

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