Western Blot Analysis of NSCLC Proteins

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.