Ab40754

The RIPA lysis buffer was purchased from Solarbio (Beijing, China) to lyse the NSCLC cells/tissues and extract the total protein, according to the experimental procedures recorded in the previous publications [36 (link), 37 (link)], the expression levels of GOT1, β-actin, cyclin D1, CDK2, cleaved caspase-3 and Bax were examined by using the Western Blot analysis. Specifically, the 40 μg/lane protein lysates were separated by using the 10 -15% SDS-PAGE, and the target protein bands were transferred onto the PVDF membranes (Millipore, USA). Next, the membranes were incubated with 5% skim milk for 70 min at room temperature for blocking, and the membranes were probed with the primary antibodies against GOT1 (1:1500, MW: 50 kDa, #PA5–24634, Thermo, USA), β-actin (1:2000, MW: 42 kDa, #ab6276, Abcam, UK), cyclin D1 (1:1500, MW: 35 kDa, #ab40754, Abcam, UK), CDK2 (1:2000, MW: 33 kDa, #ab32147, Abcam, UK), cleaved caspase-3 (1:1500, MW: 17 kDa, #ab32042, Abcam, UK) and Bax (1:1500, MW: 21 kDa, #ab32503, Abcam, UK) overnight at 4 °C. After washing by PBS buffer for 3 times, the PVDF membranes were incubated with the secondary antibody (Abcam, UK) for 120 min at room temperature. Finally, the protein bands were visualized by ECL system (GE Healthcare Bio-science, USA) and quantified by using the Image J software.

All protein was extracted in radioimmunoprecipitation assay (RIPA) buffer (Solarbio Life Science, Beijing, China) mixed with protease inhibitor on ice-cold plates. Then, the protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit purchased from Solarbio Life Science. Next, we loaded equal amounts of protein into a 10% SDS–PAGE gel for separation and transferred the protein onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% BSA (Sigma-Aldrich) and incubated with primary antibodies at 4 °C overnight. On the second day, the membranes were washed with 1× TBS solution and incubated with secondary antibody conjugated with horseradish peroxidase (HRP) at normal temperature for 1 h. Finally, the results were obtained using a ChemiDoc^TM Imaging System (Bio-Rad, Hercules, CA, USA). Throughout the entire study, primary antibodies targeting the following proteins were used: RNF31 (ab46332, 1:1000, Abcam), p53 (ab1101, 1:1000 Abcam), PCNA (ab29, 1:1000, Abcam), Cyclin D1 (ab40754, 1:5000, Abcam), Bcl-xl (2764, 1:1000, CST), GAPDH (1:1000, TransGen Biotech, Beijing), Flag-tag (D6W5B, CST), HA-tag (C29F4, CST), and β-actin (1:1000, TransGen Biotech, Beijing). The secondary antibodies were purchased from TransGen Biotech (Beijing).