Anti cdk6

Total proteins were extracted from cells utilizing Radioimmunoprecipitation Assay (RIPA) lysis buffer (Solarbio, China). Protein concentrations were then ascertained through the Bicinchoninic Acid (BCA) protein assay (Solarbio, China). Electrophoresis was performed on SDS–polyacrylamide gels, followed by transfer to PVDF membranes (Millipore, Carrigtwohill, Ireland). The membranes were then blocked with skim milk and incubated overnight at 4 °C with primary antibodies. After washing, a secondary antibody was applied and incubated at room temperature for 2 h. The antibodies used were anti-BLM (1:900 dilution; Bios, China), anti-Bax (1:5,000 dilution; Proteintech, USA), anti-PCNA (1:3,000 dilution; Proteintech, USA), anti-CDK6 (1:5,000 dilution; Proteintech, USA), anti-Cyclin D1 (1:5,000 dilution; Proteintech, USA), anti-Bcl-2 (1:2000 dilution; Proteintech, USA), anti-Cyclin E1 (1:1000 dilution; Proteintech, USA), anti-CDK2 (1:1000 dilution; Proteintech, USA), GAPDH (1:1000 dilution; Proteintech, USA), and goat-anti-rabbit, goat-anti-mouse secondary antibody (1:1,0000 dilution; Proteintech, USA). Subsequently, the results were quantified and images were processed using ImageJ software.

The total protein was extracted from A549 cells using RIPA buffer containing protease inhibitors and separated through Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Then, proteins were transferred to a PVDF membrane. After blocked with 5% fat‐free milk for 1 hour, the sample was incubated with primary antibodies for 1 hour and then with secondary antibodies for 1 hour. The following primary antibodies, namely anti‐Syncytin 1 (1:500), anti‐SP1 (1:1000) anti‐Active‐Caspase3 (1:1000), and anti‐Active‐Caspase9 (1:1000) were purchased from Abcam; anti‐Cyclin D1 (1:1000), anti‐Nusap1 (1:1000), anti‐CDK6 (1:1000), anti‐CDK4 (1:1000), anti‐Bcl2 (1:1000), anti‐Bax (1:1000), anti‐β‐catenin (1:1000), anti‐Vimentin (1:1000), anti‐E‐cadherin (1:1000), anti‐N‐cadherin (1:1000), anti‐P70 (1:1000), and anti‐GAPDH (1:5000) were purchased from Proteintech Group; anti‐p‐AKT (1:1000), anti‐p‐mTOR (1:1000), and anti‐p‐Erk1/2 (1:1000) were purchased from Cell Signaling Technology.

Total proteins were extracted by lysing cells in 6‐well plate with 60 μL RIPA buffer supplemented with protease inhibitors (#G2006, Wuhan Goodbio Technology) and phosphatase inhibitors (#G2007, Wuhan Goodbio Technology) and quantified by BCA protein assay kit (#P0011, Beyotime). One microgram of proteins was separated by 10% SDS‐PAGE and transferred to PVDF membrane (#IPVH00010, Merck Millipore). The membrane was blocked with 5% Non‐Fat Milk (#A600669, Sango Biotech) for 1 hour at room temperature and incubated with specific antibody at 4°C overnight followed by HRP‐conjugated secondary antibody incubation for 1 hour at room temperature. Images of membrane with proteins were taken with imager (Bio‐rad ChemiDoc MP, Bio‐rad). The antibodies are recorded: anti‐GAPDH (#60004‐1‐lg, Proteintech), anti‐YTHDF2 (#24744‐1‐AP, Proteintech), anti‐E‐cadherin (#20874‐1‐AP, Proteintech), anti‐N‐cadherin (#66219‐1‐AP, Proteintech), anti‐VIMENTIN (#10366‐1‐AP, Proteintech), anti‐MMP9 (#10375‐2‐AP, Proteintech), anti‐MMP2 (#10373‐2‐AP, Proteintech), anti‐SNAIL (#13099‐1‐AP, Proteintech), anti‐CDK6 (#14052‐1‐AP, Proteintech), anti‐CCND1 (#26939‐1‐AP, Proteintech), anti‐CDK4 (#11026‐1‐AP, Proteintech), anti‐KLF4 (#12173S, Cell Signaling Technology), anti‐SLUG (#C1967, Cell Signaling Technology), anti‐METTL3 (#ab195352, Abcam) and anti‐SETD7 (#ab14820, Abcam).

To isolate cellular proteins, cells were lysed with RIPA lysis buffer (Beyotime, Jiangsu, China), protein concentration was quantified using a TaKaRa BCA Protein Assay Kit (Takara Bio, Kyoto, Japan). Standard western blot procedures were performed as described previously (16 (link)), and the immunoreactive signal was exposed by an Odyssey Infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The specific primary antibodies used were as follows: anti-CDK6 (1:500, #14052-1-AP, ProteinTech, Wuhan, China), anti-Cyclin E2 (1:1000, #4132, Cell Signaling Technology, MA, USA) anti-β-actin (1:1,000, #81178, Santa Cruz Biotechnology, CA, USA).