Ecl luminescence reagent

Total protein was respectively extracted from cells in different groups by radio-immunoassay assay (RIPA) lysis buffer (Beyotime, Shanghai, China), with the concentration of protein quantified by a bicinchoninic acid (BCA) detection kit (Beyotime, Shanghai, China). 30 μg protein samples in each group were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinyl fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which was then blocked with 5% skimmed milk. Then the PVDF membrane was incubated with primary antibodies anti‐HMGB1 (1:1000, ab18256, Abcam Inc., Cambridge, UK), anti‐E-cadherin (1:1000, ab40772, Abcam Inc., Cambridge, UK), anti‐N-cadherin (1:1000, ab98952, Abcam Inc., Cambridge, UK) or anti-GAPDH (1:1000, ab9485, Abcam Inc., Cambridge, UK) overnight at 4°C, and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:2000, ab150077, Abcam Inc., Cambridge, UK) at ambient temperature for 1 h, and finally the ECL luminescence reagent (ThermoFisher Scientific, Waltham, MA, USA) was employed to detect the protein bands.

Lipofectamine™ 3000 transfection reagent was obtained from Invitrogen (Carlsbad, USA). Cell counting kit-8 (CCK-8) was provided by Dojindo (Kumamoto, Japan). anti-ERK1ERK2 (phospho T202/Y204, SP327), anti-c-Myc (phospho S62) antibody, anti-MNK1 (phospho T385, EPR2370), anti-ERK1+ ERK2, anti-MNK1 and anti-c-Myc antibodies were obtained from Abcam (London, United Kingdom). PTPRF/LAR (E6W4X) rabbit mAb was obtained from Cell Signaling Technology (Massachusetts, United States). BCA protein concentration assay kit and 5× protein loading buffer were provided by Beijing Solarbio life sciences(Beijing, China). NucleoZol was obtained from Gene Co., Ltd (Hongkong, China). ECL luminescence reagent was obtained from Thermo Scientific (Waltham, USA). qPCR Mix was provided by PROMEGA (Madison, MA, United States).

Lung tissues from each group were lysed with 100 μl/50 ml protein lysate RIPA. Briefly, the extracted protein was quantified by bicinchoninic acid (BCA) method. A total of 10% polyacrylamide gels were used for the separation of proteins. Then, the gels were transferred to polyvinylidenefluoride membranes. After blocking with 5% Skim milk/BSA, the membrane was incubated with primary antibodies (PPARγ, 1:1000, MAB3872, Chemicon International; PTEN, 1:1000, SAB4300337, Sigma; p-PTEN, pSer³⁸⁰/pThr³⁸²/pThr³⁸³, 1:1000, SAB4300044, Sigma; Akt, 1:1000, SAB4500796, Sigma; p-Akt, pSer¹²⁴, 1:1000, SAB4503853, Sigma; GSK-3β, 1:1000, G7914, Sigma; p-GSK-3β, pTyr²⁷⁹/pTyr²¹⁶, 1:1000, G5791, Sigma; β-catenin, 1:1000, C7082, Sigma; p-β-catenin, pSer³³/pSer, 1:1000, sc-57535, Santa Cruz Biotechnology), followed by treatment with HRP-labeled secondary antibody IgG (1:2000, sc-516102, Santa Cruz Biotechnology). The GAPDH (1:10000, sc-47724, Santa Cruz Biotechnology) was used as internal control. After treatment with ECL luminescence reagent (Thermo Scientific, U.S.A.), the relative gray value was analyzed using the Quantity one (Bio-Rad, U.S.A.).

The CR-CC and CS-CC cells were collected and the total proteins were extracted by using the RIPA lysis buffer purchased from Beyotime Biotechnology (Shanghai, China). The 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was next employed to separate the target proteins according to their molecular weight. After that, the separated proteins were transferred to the poly vinylidene difluoride (PVDF) membranes and blocked by 5% skim milk solution. The membranes were probed with the primary antibodies including anti-METTL1 (1:1000, Abcam, UK), anti-β-actin (1:2000, Abcam, UK), anti-Cyclin D1 (1:1500, Abcam, UK), anti-CDK2(1:1500, Abcam, UK), anti-p27 (1:1000, Abcam, UK), anti-cleaved Caspase 3 (1:1000, Abcam, UK), anti-S100A4 (1:1000, Abcam, UK) and anti-p53 (1:1000, Abcam, UK) for 2 hours at room temperature. The secondary antibody purchased from Abcam (1:1000) was sequentially incubated with the PVDF membranes. The protein bands were visualized by using the ECL luminescence reagent (ThermoFisher Scientific, USA) and quantified by performing the Image J software (NIH, USA).

RIPA lysis buffer (Beyotime Institute of Biotechnology) was utilized to isolate the total protein from the cells and the concentration of protein was analyzed using the BCA detection kit (Beyotime Institute of Biotechnology). Sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 10% gel was utilized to separate 30 µg protein sample before the protein samples were transferred to PVDF membranes (MilliporeSigma). The PVDF membrane was blocked with 5% skimmed milk for 1.5 h at room temperature and then incubated with anti-CCBE1 (1:1,000; HPA041361; MilliporeSigma), anti-Ki67 (1:1,000; cat. no. ab16667; Abcam), anti-c-Myc (1:1,000; cat. no. ab32072; Abcam), anti-cyclin D1 (1:1,000; cat. no. ab134175; Abcam) and anti-GAPDH (1:1,000; cat. no. ab9485; Abcam) overnight at 4°C and then the membrane was incubated at room temperature for 1 h with the horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. ab150077; Abcam). To identify protein bands, the ECL luminescence reagent (Thermo Fisher Scientific, Inc.) was utilized. Semi-quantitative analysis was conducted using ImageJ software (version 1.8.0; National Institutes of Health).