Nuclear and cytoplasmic protein extraction kit

Equal amounts of protein samples (28 μg) were subjected to a SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk in TBST for 2 h followed by incubation with the following primary antibodies as follows: anti-p-NF-κB P65 (1:1000, Cell Signaling Technology), anti-NF-κB P65 (1:1000, Cell Signaling Technology), anti-Iba-1 (1:1000, Abcam), anti-β-actin (1:7500, Bioss antibodies), anti-GR (1:1000, Bioss antibodies), anti-NLRP3 (1:1500), anti-ASC (1:500), anti-pro-caspase 1 (1:500), anti-caspase-1 p20 (1:750), anti-pro-IL-1β (1:10000), anti-IL-1β (1:750), anti-IL-18 (1:1000) and anti-Lamin B (1:750) both from Wanlei Biotechnology (Shenyang, China). After three times washing with TBST, the membranes were incubated with appropriate secondary antibody. The protein bands were visualized by the ECL detection system (Thermo Scientific, Waltham, MA, United States) and quantified using Image J software. Nuclear and Cytoplasmic Protein Extraction Kit (Applygen Technologies, Co., Ltd., Beijing, China) was used to extract the cytoplasmic/nuclear proteins of NF-κB P65 according to the manufacturer’s protocol.

LX-2 cells left untreated or treated with rSjp40 (5 μg/ml) for 48 h were harvested and separated using the Nuclear and Cytoplasmic Protein Extraction kit (ApplyGen, Beijing, China) according to the manufacturer’s instructions.

Cells were collected and rinsed twice with PBS and lysed with cells lysis buffer. The isolation of cytosolic and nuclear proteins was performed using nuclear and cytoplasmic protein extraction kit purchased from Applygen Technologies Inc. (Beijing, China). Protein extracts were quantified using a BCA kit purchased from Beyotime Institute of Biotechnology (Shanghai, China). Equal quantities of protein samples were loaded onto SDS-PAGE gels and transferred onto PVDF membranes (Millipore). After blocking with 5% non-fat milk, the membranes were incubated overnight at 4°C with primary antibodies in TBST. The following day, after extensive washing in TBST, the membranes were incubated with secondary antibodies. Immunoreactive bands of interest were scanned using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA).