Rabbit anti mlkl

Proteins from lung tissues and THP-1 cells were extracted and analyzed by western blotting as described previously [12 (link)]. Total proteins were extracted with cell lysis buffer (Cell Signaling Technology, USA) according to the manufacturer’s instructions. Nuclear and cytosolic proteins were obtained with a commercial nuclear extraction kit (Thermo, USA) according to the manufacturer’s instructions. The primary antibodies used in this study included: mouse anti-NLRP3, mouse anti-caspase-1 (p20) (both from AdipoGen, San Diego, CA, USA), rabbit anti-RIP3, mouse anti-p-RIP3, rabbit anti-MLKL, rabbit anti-p-MLKL (all from Abcam, Cambridge, UK), mouse anti-RIP1 (R&D Systems, Minneapolis, MN, USA), rabbit anti-phospho-p44/42 MAPK kinase (p-ERK), total p44/42 MAPK kinase (t-ERK) and fibrillarin (all from Cell Signaling Technology, Beverly, MA, USA), and rabbit anti-GAPDH and anti-NF-κB p65 antibodies (both from Santa Cruz Biotechnology, Dallas, Texas, USA). HRP conjugated anti-mouse and anti-Rabbit IgG (both from Cell Signaling Technology, Beverly, MA, USA) were used as secondary antibodies. Signals were detected with enhanced chemiluminescence analysis kit (Cell Signaling Technology, Beverly, MA, USA).

Isolated intestines were homogenized using a Mini-Rotor (Thermo Scientific). Tissues were lysed and protein concentrations were quantified. The proteins were separated by SDS-PAGE gels and were detected using chemiluminescent substrate (Thermo Scientific). Antibodies for western blotting used included: rabbit anti-MLKL (#ab184718, Abcam, 1:1000), rabbit anti-phosphor-STAT3 (Tyr 705) (#9145, Cell Signaling Technology, CST, 1:1000), mouse anti-STAT3 (#9139, CST, 1:1000), rabbit anti-PCNA (#13110, CST, 1:1000), rabbit anti-Cleaved-Caspase-3 (#9661, CST, 1:1000), rabbit anti-Cyclin D1 (#2978, CST, 1:1000), rabbit anti-C-myc (#9402, CST, 1:1000), and rabbit anti-GAPDH (#GB11002, Wuhan Service Bio Technology Co., Ltd., 1:5000).