Ab32049

Total protein was isolated using the Radioimmunoprecipitation Assay Kit (R0010, Beijing Solabio Life Sciences, Beijing, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis kit was used to prepare 10% separation gel and 5% concentrating gel samples. After electrophoresis, the protein was transferred onto a nitrocellulose membrane. The membrane was incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal antibody to TPX2 (ab71816, 1:100, Abcam), p53 (ab32049, 1:1,000, Abcam), p21 (ab47300, 1:500, Abcam), MDM2 (ab38618, 1:1,000, Abcam), bax (ab53154, 1:500, Abcam), bcl-2 (ab59348, 1:1,000, Abcam), and PCNA (ab152112, 1:500, Abcam). The membrane was rinsed three times using Tris-buffered saline containing Tween 20 on the following day. The secondary rabbit polyclonal antibody (ab7312, Abcam) was added and incubated for 1 h. Developer (D-90G, Shanghai Yingdian Detection Equipment, Shanghai, China) was added, and images were acquired using the Bio-Rad gel imaging system (Beijing Thmoregan Biological Technology, Beijing, China). IPP7.0 software (Media Cybernetics, Bethesda, MD, USA) was adopted for quantitative analysis.

NSCLC tissues from patients were fixed using 10% formaldehyde for 30 at 37°C, washed with PBS (0.01 mmol/l, pH 7.4) and followed with embedding in paraffin wax. Tissues were deparaffinized in xylene and rehydrated in grade alcohols. Tissues were cut into 4-µm thick sections and antigen retrieval was performed using Antigen Retrieval Reagents (cat. no. CTS015; Bio-Rad Laboratories, Inc.). The sections were washed with PBS for 10–15 min at 37°C and subsequently blocked using 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 2 h at 37°C. Tumor sections were incubated with CD4 (1:1,000 dilutions, Clone 4B12; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and CD8 (1:1,000 dilutions, Clone C8/144B; Dako; Agilent Technologies, Inc.), P53 (1:500 dilutions; ab32049), VEGFR (1:500 dilutions; ab36844), FGFR (1:500 dilutions; ab10646), PDGFR-β (1:500 dilutions, ab220745; all from Abcam) for 12 h at 4°C. The sections were washed three times with PBS for 3 min at room temperature and were incubated with HRP-labeled secondary goat anti-rabbit antibodies (1:2,000, ab150077; Abcam). Sections were visualized using ZEISS LSM 510 confocal microscope at ×40 magnification.

Following transfection, cells and tissues were lysed for 48 h using RIPA Lysis and Extraction Buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and the protein concentration was measured using Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Following heating at 100°C for 10 min in the presence of a loading buffer, equal amounts of protein lysates (50 µg) were separated using 10% SDS-PAGE (Bio-West Inc., Logan, UT, USA) at 100 V for 1 h, and transferred onto Invitrogen nitrocellulose membranes (Thermo Fisher Scientific, Inc.) at 120 V for 1 h. Following blocking with 5% skimmed milk (diluted with PBS), the membranes were incubated overnight at 4°C with the following primary antibodies: CYP27A1 (ab126785; 1:500; Abcam, Cambridge, MA, USA), c-myc (ab32072; 1:500; Abcam), RB (ab181616; 1:500; Abcam), Ki-67 (ab15580; 1:500; Abcam), CDK2 (ab32147; 1:500; Abcam), p21 (ab109520; 1:500; Abcam), p53 (ab32049; 1:500; Abcam), PDCD4 (ab51495; 1:500; Abcam), SOX2 (ab92494; 1:500; Abcam), β-actin (ab8227; 1:500; Abcam). Subsequently, the membranes were incubated with secondary goat monoclonal (RMG01) to rabbit IgG Fab region (Biotinylated; ab222772; 1:10,000; Abcam) at room temperature for 2 h, and proteins were detected using enhanced chemiluminescence (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).

Block paraffin samples were cut 3 µm in thickness to analyze the expression of vimentin and p53 mutant by IHC. Anti-vimentin monoclonal antibody (PRM 312 AA, dilution 1:50, Biocare Medical) was used to detect tumor expression of vimentin. Anti-p53 mutant antibody (ab32049, dilution 1:1000, Abcam) was used to detect tumor expression of p53 mutant. Vimentin and p53 mutant were considered positive if their expressions were detected in more than 10% of the tumor cells. Immunohistochemistry examination was performed using ImageJ software by a senior pathologist, blinded to the patient’s survival status and clinical data, including which chemotherapy regimen was received.