P mtor

Cells were washed twice in 2 mL ice-cold PBS and harvested with a rubber policeman after being lysed on ice for 20 min in lysis buffer (Beyotime, Shanghai, China). Equal amounts of total protein were solubilized by sodium dodecyl sulfate (SDS) sample buffer (Beyotime, Shanghai, China), separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Membranes were incubated with corresponding polyclonal and secondary antibodies. Signal was detected with a Super ECL Reagent substrate (Hai Gene, Harbin, China). Source and product number of antibody: LC3B (Abcam, ab48394), SQSTM1/p62 (Abcam, ab101266), Beclin 1 (Bioworld, AP0769), p-ULK1 (Affinity Biosciences, AF4387), ULK1 (Proteintech, 20986-1-AP), p-ATG13 (Bioworld, BZ40743), ATG13 (Bioworld, bs6045), ATG5 (Proteintech, 10181-2-AP), p-PI3K (CST, 4228), PI3K (CST, 4249), p-PTEN (CST, 9554), PTEN (CST, 9188), p-Akt (CST, 13038), Akt (CST, 4691), p-mTOR (Abcam, ab84400), mTOR (CST, 2972S), p-p70 S6K (CST, 9205), p70 S6K (CST, 2708), p-4E-BP1 (CST, 2855), 4E-BP1 (CST, 9644), GAPDH (Bioworld, AP0066), HRP-linked antibody (anti-rabbit IgG, CST, 7074), Goat Anti-Rabbit (Alexa Fluor®488) (Abcam, ab150077), goat anti-rabbit (Alexa Fluor®647) (Abcam, ab150079).

The total cellular protein was isolated using RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors (Sigma-Aldrich, USA). Equal amounts of total proteins were loaded on SDS-PAGE gels for separation of proteins according to their molecular weights and then transferred onto PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 and then incubated with primary antibodies overnight at 4 °C, followed by incubation with corresponding secondary antibodies at room temperature for 2 h. The protein bands were visualized using enhanced chemiluminescence. Immune response bands were exposed on a Bio-Rad ChemiDoe XRS + Imaging System (Bio-Rad, USA). The primary antibodies used were NRP1 (Abcom, cat# ab81321), EGFR (CST, cat# 54359), p-EGFR (CST, cat# 2234), CDK2 (Abcam, cat# ab32147), CDK4 (CST, cat# 12709), CDK6 (CST, cat# 13331), CyclinD1 (CST, cat# 2922), E-cadherin (CST, cat# 3195S), N-cadherin (CST, cat# 13116S), AKT (CST, cat# 4691L), p-AKT (CST, cat# 4060L), GSK3β (CST, cat# 12456S), p-GSK3β (CST, cat# 5558S), mTOR (Abcam, cat# ab32028), p-mTOR (Abcam, cat# ab109268), FLAG (F1804, Sigma), HA (TA180128, OriGene), and GAPDH (Santa Cruz, cat# sc-365062).

The treated A549 or H1299 cells were collected, washed with PBS and lysed in RIPA buffer (Beyotime, Shanghai, China). The protein concentration was quantified using a Protein BCA Assay Kit (Bio-Rad, Hercules, California, USA). Equal amount of protein samples (30 μg) was fractionated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% skim milk in 0.05% TBS-Tween-20 (v/v) for 1 h, the membranes were incubated overnight at 4 °C with the primary antibodies against PKM2 (Sigma), phosphorylated mammalian target of rapamycin (p-mTOR) (Abcam, Cambridge, UK), mTOR (Abcam), or β-actin (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with horseradish peroxidase (HRP)-linked secondary antibody (Abcam) for 2 h. The signal bands were visualized by an electrochemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA).

Protein extraction was performed by lysing the cells in RIPA (radio-immunoprecipitation assay) buffer. The protein concentration was determined using the DC protein assay, and equal amounts of protein were loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked with 5% nonfat milk in TBST for 1 h. The primary antibodies were added and incubated overnight at 4 °C with shaking. The membranes were then washed and incubated with secondary antibodies for 1 h at room temperature. The primary antibodies of TTl1, FGFR3, α-actin, p53, p21, ATF3, FAS, TOM20, mtTFA, and PGC1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); TELO2, Cyclin D1, and γ-H2A.x were obtained from Abcam (Cambridge, UK); and p-mTOR, mTOR, p-ATM, ATM, p-p53 (ser15), MDM2, DEC1, p-c-JUN, c-JUN, p-Akt, Akt, p-p38, p38, p-PI3K, PI3K, p-ERK, ERK, p-Chk2, Chk2, p-AMPK, AMPK, p-eIF2α eIF2α, p-P70S6K, P70S6K, p-TSC2, TSC2, and 4EBP1 were obtained from Cell Signaling (Danvers, MA, USA).

Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were obtained from Gibco. Earle's balanced salt solution (EBSS) and propranolol were purchased from Sigma‐Aldrich. Rapamycin (RAPA), 3‐methyladenine (3‐MA) and chloroquine (CQ) were obtained from MedChemExpress, and ZVAD‐FMK, necrostatin‐1, liproxstatin‐1, SB203580, SP600125, SC79 and 740Y‐P were purchased from SelleckChem. Then, these reagents were dissolved in dimethyl sulfoxide (DMSO) (MP Biomedicals or water and stored at −80°C. Primary antibodies against Akt, p‐Akt, p38 MAPK, p‐p38 MAPK, JNK, p‐JNK, Erk1/2 and p‐Erk1/2 were purchased from Cell Signalling Technology. Primary antibodies against alpha‐smooth muscle actin (α‐SMA), fibronectin (FN), LC3B, P62, p‐PI3K p85, PI3K p85, ATG9b, ATG9a, mTOR, p‐mTOR, ATG12, ATG5 and anti‐ubiquitin were procured from Abcam. Primary antibodies against beta‐actin, alpha‐tubulin, GAPDH and HRP‐conjugated secondary antibodies were obtained from Proteintech. DyLight 549‐conjugated and DyLight 488‐conjugated secondary antibodies were provided by Abbkine.

Extracellular matrix (ECM) media were purchased from ScienCell. RPMI-1640 media was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Trizol was purchased from Sigma-Aldrich (Sigma-Aldrich, Burlington, MA, USA). Hiscript QRT Supermix for quantitative PCR (qPCR) and AceQ qPCR SYBR Green was purchased from Vazyme (Nanjing, China). Antibodies (TRIM22, Beclin1, P62, ATG7, ATG5, mTOR, P-mTOR, AMPK, P-AMPK, ERK, P-ERK, and GAPDH) were purchased from Abcam (Abcam, Cambridge, UK). Age I and EcoRI were purchased from NEB (New England Biolabs, Ipswich, MA, USA). The BCA Protein Assay Kit was purchased from HyClone-Pierce, Inc (Thermo Fisher Scientific). Real-time fluorescence quantitative polymerase chain reaction (PCR) was purchased from ABI (StepOne PLUS, Thermo Fisher Scientific). The gel imager was purchased from Bio-Rad (Bio-Rad, Hercules, CA, USA). The ultraviolet–visible spectrophotometer was purchased from Thermo Fisher Scientific. The chemiluminescence imaging system used was a GE AI600. The microplate reader was purchased from Bio-Rad.

Cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche) for 30 min and then centrifuged to remove the Triton-insoluble pellet. Samples normalized with equal protein concentration were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. Polyvinylidene fluoride (PVDF) membranes were incubated with the following primary antibodies for overnight at 4°C: UBQLN1 (Abcam, 1:2000), p-mTOR (Abcam, 1:2000), mTOR (Abcam, 1:2000), p-p70S6K (Abcam, 1:2000), p70S6K (Abcam, 1:2000), GAPDH (Sigma, 1:1000). Goat anti-mouse (1:5000) or anti-rabbit (1:5000) secondary antibodies (BBI Life Science Corp.) were used. The protein bands were visualized using chemiluminescence imaging system V1.8.0.112 (Tannon Science & Technology Co., Ltd,). All experiments were independently performed in triplicates [19 ].