Ab171030

The cell protein was extracted to determine its concentration according to instructions of BCA protein assay kit (Pierce, Thermo Fisher Scientific, USA), followed by adding loading buffer for 10 min boiling at 95 °C. After loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the total protein of 60 μg was transferred to polyvinylidene fluoride (PVDF) membrane and sealed with 5% bovine serum albumin (BSA) at room temperature for 1 h. Next, PVDF membrane was incubated with anti-SAA1 (ab171030, 1 μg/ml), anti-NADPH oxidase 4 (ab133303, 1/2000), anti-phospho-p38 (ab4822, 1/1000), anti-p38 (ab197348, 1/500), anti-phospho-NF-kB p65 (ab86299, 0.04 μg/ml), anti-NF-kB p65 (ab16502, 1 μg/ml) and β-actin (ab227387, 1/20000) (all purchased from Abcam, Cambridge, MA, USA) at 4 °C overnight. With tris buffered saline with Tween (TBST) washing 3 times/5 min, PVDF membrane was incubated with goat anti-rabbit IgG H&L (HRP) (ab6721, 1/5000, Abcam, Cambridge, MA, USA) for 1 h and washed 3 times/5 min. Taking β-actin as loading control, PVDF membrane was developed by chemiluminescent reagent using Bio-rad Gel Dol EZ imager (GEL DOC EZ IMAGER, Bio-rad, California, USA). The gray value of target band was analyzed using Image J software. The experiment was repeatedly carried out in triple.