Anti atg14

WB and IHC were performed according to established methods in the previous study [38 (link)]. Primary antibodies were used at appropriate dilution concentration as follows: WB, anti-ATG13 (Protein Tech, Chicago, IL, USA, 1:2000), anti-ATG14 (Protein Tech, 1:1000), anti-SQSTM1/p62 (Protein Tech, 1:4000), anti-BECN1 (Abcam, Cambridge, UK, 1:1000), anti-LC3B (Protein Tech, 1:2000) and β-actin (Abcam, 1:2500). IHC, anti-BECN1 (Abcam, 1:100).

Homogenized tissues and cells were lysed in RIPA buffer containing 1 × PBS, 1% NP-40, 5 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na₃VO₄, 1% phenylmethanesulfonyl fluoride, complete protease inhibitor cocktail (Sigma) and complete phosphatase inhibitors. The lysates were centrifuged at 12,000 g for 10 min at 4°C to remove the insoluble materials, and the supernatants were boiled in SDS loading buffer. The boiled samples were separated by 10% SDS–polyacrylamide gel and electroblotted to nitrocellulose transfer membranes (Whatman, GE Healthcare). The membranes were blocked with 5% milk and incubated with different antibodies, followed by incubation with fluorescent-conjugated secondary antibodies. The primary antibodies used in western blotting included anti-p-IRβ [tyr1162/1163], anti-IRβ (Santa Cruz, Dallas, TX, USA), anti-p-AKT [ser473], anti-AKT, anti-BECLIN1, anti-LC3I/II, anti-p-GSK3β [ser9], anti-GSK3β, and anti-β-actin (Cell Signaling, Beverly, MA, USA) and anti-ATG14 (Proteintech, Chicago, IL, USA).