Imagej lab software

Whole cell lysates were isolated using protein extract buffer, and tumor proteins were isolated using RIPA lysates (Solarbio, Beijing, China) with phenylmethylsulfonyl fluoride (PMSF) in the cocktail. Equal amounts of total protein were electrophoresed using SDS/PAGE. Protein signals were visualized using chemiluminescence reagents. Protein concentrations were estimated by the BCA method. The protein expression was analyzed by ImageJ Lab software (Bio-Rad, Hercules, CA, USA).

Tissue proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (ATTO, Tokyo, Japan) with proteinase and phosphatase inhibitors (ATTO, Tokyo, Japan). Protein concentration was measured using a bicinchoninic acid (BCA) assay (Thermo Scientific, Waltham, MA, USA). The proteins (20 µg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Pall, Port Washington, NY, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween (TBST) buffer for 1 h at 4 °C and incubated with primary antibody diluted in TBST at 4 °C overnight. The membranes were rinsed with TBST buffer and incubated with horseradish peroxidase (HRP)-labeled secondary antibody diluted in TBSTfor 1 h. The antibodies are presented in Supplementary Table S3. Next, the membranes were incubated with West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA). Protein expression was detected by exposure to X-ray film (Agfa, Mortsel, Belgium) and analyzed using ImageJ lab software (Bio-Rad, Hercules, CA, USA).