Enhanced chemiluminescent autoradiography

Total protein was extracted using a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 1 mM EDTA, pH 7.5) containing a Complete protein inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) to generate the whole protein lysate following centrifugation at 14,000 × g at 4°C for 15 min. An equal amount of 50 µg protein/lane was loaded into each lane in a 12% SDS-PAGE gel. The proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane by electrophoresis. Following blocking using 5% milk in Tris-buffered saline with 0.1% Tween for 1 h at 25°C, the membranes were incubated with primary antibodies against Trim59 (1:1,000; catalog no. ab166793; Abcam, Cambridge, UK) overnight at 4°C. A secondary antibody conjugated with horseradish peroxidase (goat-anti rabbit; 1:5,000; catalog no. SC-2030; Santa Cruz Biotechnology Inc.) that recognizes the primary antibody was then added at room temperature for 1 h, and the immunoreactivity was determined with enhanced chemiluminescent autoradiography (Thermo Fisher Scientific, Inc.). β-actin was used as a loading control. Each experiment was repeated at least three times.

Human lung cancer cell lines H-125, NCI-H292, H1688, NCI-H446, and H1975 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in recommended media (Gibco, Los Angeles, CA, USA) supplied with 10% fetal bovine serum (FBS) (Gibco). Lung cancer cells 95D and A549, and normal lung cell line MRC-5 were obtained from the Cell Bank of the Chinese Academy of Sciences and cultured at a 37°C incubator supplied with 5% CO₂. Proteins from NCI-H929 and H1688 cells were extracted for subsequent immunoblot analysis when the growth confluence of siuc.338-treated cells was above 80%. Primary antibodies against cyclin B1, Cdc25C, and vimentin were commercially from Abcam (Cambridge, UK). Primary antibodies against Snail, N-cadherin, E-cadherin, GAPDH, and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Immunoreactivities were determined with enhanced chemiluminescent autoradiography (Thermo Scientific) with the machine Las3000.

Total protein lysates were obtained from cultured cells with lysis buffer supplemented with protease inhibitors. After determination of the qualities and concentrations of each extraction with BCA protein assay kit (Beyotime, Nanjing, People’s Republic of China), 50 μg whole proteins were loaded onto a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (Invitrogen). Afterward, proteins were transferred to nitrocellulose membrane, and nonspecific bindings were eliminated with 5% milk/TBST. Following overnight incubation with primary antibodies at 4°C, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at 37°C. Immunoreactivity was detected with enhanced chemiluminescent autoradiography (Thermo Scientific). Primary antibodies against SPOCK1, Cdc25C, cyclin D1, cyclin B1, GAPDH, and secondary antibodies were commercially purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against PI3K, AKT, Bad, Bcl-xL, Bcl-2, and antibodies for detection of phosphorylated PI3K (p-PI3K) at Y607 and phosphorylated AKT (p-AKT) at Ser473 were bought from Abcam (Cambridge, MA, USA). Matrix metalloproteinase 3 (MMP3) and MMP9 antibodies were purchased from Cell Signaling Technology (Danver, MA, USA).