P mtor

Western blot was performed as previously described [28 (link)]. Antibodies used were the following: β-actin (1:2000, Cat# GB15003, Servicebio), ANXA2 (1:1000, Cat# F0921, Santa Cruz), TTK (1:1000, Cat# A300-296A, Bethyl), Akt (1:3000, Cat# 4691 T, CST), p-Akt (1:3000, Cat# 4060 T, CST), β-Catenin (1:3000, Cat# 8480 T, CST), Snail (1:3000, Cat# 3879 T, CST), Claudin-1 (1:3000, Cat# 13255 T, CST), mTOR (1:3000, Cat# T55306, Abmart), p-mTOR (1:3000, Cat# T5657, Abmart), Myc-tag (1:3000, Cat# 1:5000, Abmart), and Anti-Flag-tag (1:2000, Cat# A5712, Selleck). An ECL Enhanced Kit (Cat# B520A, Biosharp, China) was used to visualize protein-antibody complexes.

Total protein was extracted from cell samples using RIPA lysis buffer (Beyotime Biotechnology) supplemented with Thermo Scientific Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Protein extracts were resolved by SDS-PAGE and then electrophoretically transferred onto a PVDF membrane (EMD Millipore). The membrane was incubated for 2 h in blocking buffer (1 × TBST containing 5% non-fat milk), and then was incubated at 4℃ overnight with following primary antibodies: MIAC (abmart, 1:1000), AQP2 (abcam, ab199975), EREG (Cell Signaling, 12,048), EGFR (Proteintech, 18,986–1-AP), Phospho-EGFR (Abmart, T55232), mTOR(Abmart, T55306), p-mTOR (Abmart, T56571), Akt (Abmart, T55561), Phospho-Akt (Abmart, T40067), ERK1/2 (Abmart, T40071), Phospho-ERK1/2 (Abmart, TP56192), GAPDH (Proteintech, 60,004–1-Ig), HA (Abmart, M20003S). The immunocomplexes were subsequently incubated with the HRP-conjugated secondary antibodies, and detected with the Western Blot Detection kit (Beyotime Biotechnology) and TANON-5200 system (Tanon, Shanghai, P.R. China). The densitometric ratio of protein bands was calculated by ImageJ program.

Total protein was extracted from mouse lung tissues and cells using a prepared SDS lysis buffer for protein blotting analysis. The proteins were separated on SDS–polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Germany). The membrane was then blocked with 5% skimmed milk for 2 h. Antibodies against β-actin (Abcam, #ab8226), Collagen I (Arigo Biolaboratories, #ARG21965), TGF-β1 (ImmunoWay, #YT4632), P-NFKB (Wanleibio, #WL02169), NFKB (Bioss, #bsm-33117M), TNF-α (Proteintech, #60291-1-Ig), IL-1β (Wanleibio, #WLH3903), HIF-1a (Proteintech, #20960-1-AP), SOD1 (Wanleibio, #WL01846), SOD2 (Boster, #BA4566), LC3-II (Cell Signaling Technology, #12741), P62 (Cell Signaling Technology, #16177S), LAMP2 (Proteintech, #66301-1-Ig), P-mTOR (Abmart, #T56571), mTOR (Abmart, #T55306), P-ULK1 (ImmunoWay, #YP1544), ULK1 (Abmart, #T56902), Beclin1 (Boster, #PB0014), Atg5 (Bioss, #bs-4005r), BNIP3 (Santa Cruz Biotechnology, #sc-56167), P-AKT (Abmart, #T40067), AKT (Abmart, #BM4400), and TMEM175 (Proteintech, #19925-1-AP) were used. The PVDF membrane was incubated with these antibodies at 4 °C overnight. Subsequently, an HRP-labeled secondary antibody (ZSGB-BIO, Beijing, China) was added and incubated for 1 h, followed by detection using the ECL luminescence system.