Ripa cell lysis buffer

The cells were lysed with the RIPA cell lysis buffer containing protease and phosphatase inhibitors (Roche, Germany). The total protein content in the lysates was determined using the BCA kit (Beyotime Biotechnology, China). Equal amounts of protein per sample were separated on a 10% sodium dodecyl sulfate-polyacrylamide (SDS-PA) gel, and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk, the membranes were incubated overnight with the primary antibodies (ABclonal, China) at 4 °C. The membrane was washed thrice and incubated with the HRP-conjugated secondary antibody (ABclonal, China) for 1 h. The positive bands were visualized using Clarity Western ECL Substrate (Bio-Rad, USA).

RAW264.7cells were stimulated with rLsa21 and cells were recovered at various time points and treated with RIPA cell lysis buffer supplemented with proteinase and phosphatase inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). The cells were centrifuged at 4 °C, 12,000 × g, for 30 min, and protein concentrations were measured using the BCA protein-quantification assay. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to PVDF membranes (Millipore, Bedford, MA). After blocking with 5% nonfat milk in PBS (pH 7.4, 0.05% (v/v) Tween-20), the membranes were incubated overnight at 4 °C with primary Abs, including rabbit anti-mouse-p38, anti–mouse phospho-p38, anti-mouse-ERK2, anti–mouse phospho-ERK1/2, anti–mouse stress-activated protein kinase/JNK, anti–mouse phospho–stress-activated protein kinase/JNK, anti-mouse IκBα (Cell Signaling Technology, Beverly, MA), according to the manufacturer’s instructions. After washing with PBS, the membranes were incubated for 1–2 h at 37 °C with the appropriate HRP-conjugated anti-rabbit IgG secondary Ab (1:5000, CST, USA). The peroxidase-positive bands were detected using an ECL detection kit.