Western blot analysis

Western blot analysis was performed to observe the expression of proteins in HL-1 cells after stimulating by mouse homologous IL-2. Cells were cultured in 9.6 cm² plates and transfected as described. After 48 h, cells were harvested and incubated in ice-cold TNEN lysis buffer (in mmol/L: 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 2.0 mM EDTA, 1.0 % Nonidet P-40) with 1 mini tab of EDTA-free protease inhibitors (Roche) and 1 mmol/L PMSF (phenylmethylsulfonyl fluoride) for 30 min at 4 °C. The insoluble fraction was pelleted by centrifugation at 12,000 x g for 15 min at 4 °C. 100 μl of supernatant was mixed with 20 μl of 6X laemmli buffer (0.3 mol/L Tris–HCl, 6%SDS, 60 % glycerol, 120 mmol/L dithiothreitol (DDT) and proprietary pink tracking dye), and heated at 37 °C for 10 min. 20 μl of samples were subjected to SDS-PAGE. Proteins were transferred onto a 0.45um polyvinylidene fluoride (PVDF) membrane (Millipore) after electrophoresis. The membrane was probed with an anti-p53 mouse monoclonal antibody (abcam, PAb240) or anti-scn3b rabbit polyclonal antibody (GeneTex, GTX104440), followed by incubation with a HRP-conjugated secondary goat anti-mouse or goat anti-rabbit antibody respectively (Millipore). The protein signal was visualized by a Super Signal West Pico Chemi luminescent substrate according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, Illinois, USA). Mouse GAPDH (Proteintech, 14C10) was used as loading control. Each assay was performed in triplicate and repeated at least three times.