Cdk2 antibody

Total proteins were extracted from the cells using RIPA buffer and a protease‐inhibitor cocktail. After electrophoresis in SDS‐PAGE, the proteins were transferred to PVDF membranes. The membranes were blocked with 10% milk and incubated with primary antibodies at 4°C overnight. The following antibodies were used: SQLE antibody (Abcam), c‐Myc antibody (Abcam), PCNA antibody (Abcam), CDK2 antibody (Abcam), cyclin D1 antibody (Abcam), β‐actin antibody (Abcam), GAPDH antibody (Abcam), p‐ERK1/2/ERK1/2 antibody (Abcam) and p‐p65/p65 antibody (Abcam). After washing with 1 × TBST for three times, the membrane was incubated with the corresponding secondary antibody.

H&E staining: the lung tissues were paraffin-embedded and cut into 45 μm thick sections. The slices were dewaxed with xylene, dehydrated with gradient alcohol, and stained with after embedding and paraffin sectioning. The slices were photographed under a light microscope (100x).
IHC: the sections were deparaffinized, rehydrated, and then subjected to antigen retrieval for 10 minutes in an antigen retrieval buffer. Next, the sections were then treated with 3% H₂O₂ for 15 minutes at room temperature to block the activity of endogenous peroxidases. After that, the sections were incubated with goat serum to prevent nonspecific binding for 15 minutes. Afterward, these sections were incubated overnight at 4°C with the MUC1 antibody (dilution: 1 : 200, Cell Signaling Technology 4538S) or the CDK2 antibody (dilution: 1 : 200, Abcam ab32147). The next day, the sections were incubated with the corresponding secondary antibodies (dilution: 1 : 500; Thermo Fisher Scientific, USA) for 1 hour at 37°C. The sections were then stained with DAB (3, 3′-diaminobenzidine) Kit (Biyotime) for 5 min and examined using a light microscope at 100x magnification.

Cells were collected in RIPA lysis buffer, and the proteins were quantified using a BCA Protein Concentration Assay Kit (MDL Biotechnology, China). The protein was isolated on a 10 % SDS-PAGE gel (MDL Biotechnology, China), and transferred onto PVDF membranes. Then, the membranes were blocked with 5 % (w/v) nonfat dry milk in PBS and 0.05 % Tween. The primary antibodies used as follows: anti-FAK antibody (1:1,000; MDL Biotechnology, China), anti-CDC27 antibody (1:1,000; MDL Biotechnology, China), RPRD1A-antibody (1:1,000; MDL Biotechnology, China), RPL34-antibody (1:1,000; MDL Biotechnology, China), RPL24-antibody (1:1,000; MDL Biotechnology, China), EIF6-antibody (1:1,000; MDL Biotechnology, China), p53-antibody (1:1000; Affinity, USA), MDM2-antibody (1:1000; Abcam, USA), p21-antibody (1:1000; Epitmics, USA), CDK2-antibody (1:3000; Abcam, USA), CDK4-antibody (1:3000; Abcam, USA), RB-antibody (1:2000; Abcam, USA), ATM-antibody (1:3000; Abcam, USA), E₂F₁-antibody (1:1000; Abcam, USA). β-actin (1:1000; MDL Biotechnology, China) or GAPDH (1:5000; Abcam, USA) was used as the loading control. The secondary antibodies were incubated with goat IgG-HRP (1:5,000; MDL Biotechnology, China) at room temperature for one hour. The blots were detected using a ChemiDoc MP chemiluminescence imaging system (Bio-rad, USA).