Nucleus protein extraction kit

Total cell lysates were extracted by using radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China). Nucleus proteins were extracted by using nucleus protein extraction kit (Beyotime, Shanghai, China). Lysates were collected and centrifuged at 12,000 rpm. Loading buffer was added to the supernatant of samples and the proteins were denatured at 100°C for 5 min. Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to PVDF membrane. The membranes were blocked with 5% non-fatted milk, washed four times with Tris-buffered saline plus Tween (TBS-T, 15 min each time), and then incubated with the following primary antibodies: p-JNK, JNK, p-Akt (Ser 473), Akt, p-PI3K, PI3K, Fas, cleaved caspase-3, 7, 8, 9, p-IKK-β, IKK-β, p-IκBα, IκBα, NFκB p65, p-ERK, ERK, p-p38, p38, poly(ADP-ribose) polymerase (PARP), Bad, Bax, Bcl-xl, Bcl-2, Lamin B, GAPDH (Cell Signaling Technology, Beverly, MA, United States). After overnight incubation at 4°C, the membranes were washed four times with TBS-T and then incubated with HRP conjugated secondary antibodies according to each species for another 2 h at room temperature. The relative band density was determined by using the Bio-Rad Imaging System (Hercules, CA, United States) with an enhanced chemiluminescence (ECL) western blotting substrate kit (Tianmen, China).

The bMECs and macrophages were lysed in buffer containing 10 mM Tris–buffer (pH 7.5), 1 mM PMSF, 1% Triton X–100, 1 mM protease inhibitor cocktail, and 1 mM phosphatase inhibitor. Nucleus extracts were prepared using a KeyGEN nucleus protein extraction kit and protein concentration was quantified using a BCA Protein Assay kit (Beyotime), according to the manufacturer’s instructions. Proteins (30–100 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and electrotransferred to PVDF membranes (Millipore Corporation, St. Charles, MO, United States) by a wet transferor (BIO-RAD, United States). Then, PVDF membranes were blocked at room temperature for 2 h in 5% non-fat dry milk in 0.1% Tween-20–Tris buffered saline, pH 7.4 (TBST). After blocking, membranes were incubated overnight with specific primary antibodies at 4°C. Thereafter, membranes were probed with HRP-conjugated secondary antibody for 1 h at room temperature and fluorescence detected with an enhanced chemiluminescence system (ECL; Beyotime). Results were normalized to GAPDH, and band density was analyzed with Image J (National Institutes of Mental Health, Bethesda, MD, United States). Each group was assayed six times (three independent experiments, with two replicates per group).