Enhanced chemi luminescence (ecl)

RIPA solution (Thermo-Fisher Scientific) was used to extract total protein. All operations were performed in strict accordance with the instructions of the kit. BSA assay was performed to measure protein concentration. Protein samples were mixed with loading buffer with a ratio of 1: 5 and were denatured at 85°C for 1 h. SDS-PAGE gel electrophoresis was performed with 35 μg protein in each well. After gel transfer to PVDF membranes, membranes were blocked with 5% skimmed milk at 25°C for 1 h, followed by incubation with rabbit anti-human primary antibodies of TGF-β1 (ab9758, 1: 1500; Abcam) and GAPDH (rabbit anti-human, ab9485, 1: 1400, Abcam) at 4°C overnight. Membranes were then incubated with IgG-HRP secondary antibody (1: 1000, MBS435036, MyBioSource) at 25°C for 2 h. ECL (Sigma-Aldrich, USA) was then used to develop signals, and the grey band of TGF-β1 was normalized to that of GAPDH using Image J software to represent relative TGF-β1 expression level.

Total protein extraction was performed using RIPA solution (Thermo Fisher Scientific, Inc.), and protein concentration was assessed using a bicinchoninic acid assay. A total of 30 µg protein from each sample was separated by 10% SDS-PAGE and then transferred to PVDF membranes. Blocking was performed by incubation with 5% skimmed milk in PBS at room temperature for 2 h. Following washing, membranes were incubated with rabbit polyclonal anti-RUNX2 (1:1,000; cat. no. ab23981) or rabbit polyclonal anti-GAPDH (1:1,000; cat. no. ab9485) primary antibodies (all Abcam) overnight at 4°C. Membranes were washed and incubated with goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:1,000; cat. no. MBS435036; MyBioSource, Inc.) at room temperature for 3 h. Following another round of washing, ECL (Sigma-Aldrich; Merck KGaA) was added to visualize the protein bands. Signals were then scanned using the MYECL™ Imager (Thermo Fisher Scientific, Inc.). Relative expression levels of each protein were normalized to those of the endogenous control β-actin using Image J v1.48 software (National Institutes of Health).

RIPA solution was used to isolate total proteins. BCA assay (Sigma-Aldrich) was used to quantify protein samples. After denaturation, proteins were separated using SDS-PAGE gels (8%). Gel transfer to PVDF membranes was performed and blocking was carried out in 5% non-fat milk. The primary antibodies were GAPDH (1:1000, Abcam) and PD-L1 (1:1000, Abcam), CDK4 (1:1000; Cell Signaling Technology), CDK6 (1:1000; Cell Signaling Technology), and β-tubulin (1:2000; Wanleibio, Beijing, China). The secondary antibody was HRP (IgG) goat antibody (1:1000; ab6721; Abcam). ECL (Sigma-Aldrich) was used to develop signals. Quantity One software was used to quantify data.

Protein lysates were collected from A549 or NCI-H1975 cells using RIPA Buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitor cocktail. Protein concentrations were estimated using bicinchoninic acid protein assay kit (CWBIO, Beijing, China) and proteins were denatured for 10min at 95°C. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with non-fat milk in Tris-buffered saline and Tween 20. After that, the membranes were incubated with primary antibodies against Bax (Abcam, ab32503, 1:5000), Bcl-2 (Abcam, ab32124, 1:1000), Cleaved caspase3 (Abcam, ab2302, 1:500), Caspase3 (Abcam, ab32150, 1:1000), PTEN (Abcam, ab32199, 1:10,000), p-PI3K (Abcam, ab182651, 1:1000), p-AKT (Abcam, ab38449, 1:1000) and GAPDH (Abcam, ab9485, 1:2000) at 4 °C overnight. On the second day, the membranes were incubated with the corresponding IgG-HRP secondary antibody (Abcam, ab205718, 1:20,000) at room temperature for 1 h. Signals were developed using ECL (Sigma-Aldrich, MO, USA) and analyzed by Image J software (NIH, USA).

Total proteins in 3×10⁴ cells of each transfection group were isolated by RIPA solution (Sangon). BCA assay (Sangon) was performed to quantify protein samples. After proteins were denatured at 95°C for 10 min, proteins were separated by performing electrophoresis (12% SDS-PAGE gel). PVDF membrane was used to transfer proteins and 5% non-fat milk (PBS) was used for blocking. After that, membranes were first incubated with anti-GAPDH (1: 1000, ab37168, Abcam) and anti-TGF-β1 (1: 1000, ab92486, Abcam) rabbit primary antibodies for 18 h at 4°C. Following that, IgG-HRP (1: 1500, MBS435036, MyBioSource) goat secondary antibody was used to further incubation for 2 h at 24°C. Finally, ECL (Sigma-Aldrich, USA) was used for signal production and all data were normalized using Image J v1.48 software.