Ccd camera image system

Total protein was extracted using RIPA buffer (Beyotime) containing a protease inhibitor (Roche). The proteins were separated by SDS‐PAGE and transferred onto PVDF membranes (Millipore). The membranes were blocked with 10% milk for 2‐4 h and incubated with primary antibodies at 4°C overnight. The primary antibodies used were as follows: anti‐ADFP (1:500; AbGene, China), anti‐p‐Akt (Ser 473) (1:1000; CST), anti‐Akt (1:1000; CST), anti‐p‐STAT3 (1:1000; Abcam), anti‐STAT3 (1:1000; Abcam), anti‐p‐ERK (1:1000; CST, anti‐ERK (1:1000; CST), anti‐β‐catenin (1:1500; Santa, anti‐NFκB (1:1500; Abcam), anti‐Bcl‐2 (1:1000; Abcam), anti‐Bax (1:1500; Abcam) and anti‐GAPDH (1:2000; Abcam). After washing in TBST, the membranes were incubated with an anti‐rabbit IgG secondary antibody (1:10 000; Solarbio) at room temperature for 2 h. Lastly, signals were detected using an HRP Chemiluminescent Kit (Millipore) and CCD camera image system (Bio‐Rad).

All 137 RB patients could divided into three groups according to the genotype at rs937283. We randomly selected 8 RB tumor tissues from each groups and then isolated protein. Frozen tumor tissues from RB patients were homogenized on ice and lysed in RIPA buffer containing protease inhibitor (cOmplete, Sigma, CA, USA) and PMSF. The homogenate were sonicated and centrifuged at 12,000 rpm at 4 °C for 5 min to remove cell debris. A BCA assay kit (Beoytime Biotech, Shanghai, China) was used to determine protein concentration and extracted proteins were separated by 8% SDS-PAGE, then separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The membrane was blocked with 5% skim milk in TBST (TBS buffer with 0.1% Tween-20), and then incubated with human MDM2 antibody (1:1000 dilution, sc-5304, Mouse Monoclonal IgG, Santa Cruz, USA), p53 antibody (1:1000 dilution, ab28, Mouse Monoclonal IgG, Abcam USA) and GAPDH antibody (1:1000 dilution, sc-32233; Mouse Monoclonal IgG, Santa Cruz, USA) at room temperature for 2 h. Subsequently, horseradish peroxidase(HRP) -conjugated anti-mouse IgG were used as secondary antibody (1:5000 Dilution, Beoytime Biotech, Shanghai, China). Signals were captured by a CCD camera image system (Bio-Rad, CA, USA) with an HRP Chemiluminescent kit (Beoytime Biotech, Shanghai, China).

Frozen tumor tissues and adjacent tissues were homogenized on ice with a glass homogenizer and lysed in RIPA buffer containing protease inhibitor (cOmplete, Sigma, CA, USA) and PMSF. Lysates were sonicated and centrifuged at 13,000 rpm at 4°C for 5 min. Then samples were precipitated, and supernatants were collected. A BCA assay kit (Beoytime Biotech, Shanghai, China) was used to measure protein concentration and extracted proteins were subjected to 8% SDS-PAGE, then separated proteins were transferred onto PVDF membranes (Millipore, MA, USA). The PVDF membrane was blocked with TBST (TBS buffer with 0.1% Tween-20) containing 5% skim milk and incubated with human CEACAM1 antibody (1ug/ml; Clone 283324; Monoclonal Mouse IgG1, R&D System, USA) and GAPDH antibody (1:1000 dilution, sc-32233; Mouse Monoclonal IgG1, Santa Cruz, USA) at room temperature for 2 h. Subsequently, samples were incubated with HRP-conjugated secondary antibody (1:5000 Dilution, Beoytime Biotech, Shanghai, China). Signals were captured using an HRP Chemiluminescent kit (Beoytime Biotech, Shanghai, China) and CCD camera image system (Bio-Rad, CA, USA).