Rabbit anti p mlkl

THP-1 cells were fixed in 4% paraformaldehyde for 10 min, permeabilized in 0.01% Triton X-100 for 10 min and blocked in 5% BSA for 1 h. For p-RIP3 and p-MLKL staining, cells were incubated with mouse anti-p-RIP3 and rabbit anti-p-MLKL (both from Abcam, Cambridge, UK), overnight at 4 °C and then labeled with secondary anti-mouse antibody conjugated with Alexa Fluor 488 and anti-rabbit antibody conjugated with Alexa Fluor A555 (both from Thermo Fisher Scientific, Grand Island, NY, USA) respectively for 60 min, then mounted in Vectorlabs mounting media with 4′,6-diamidino-2-phenylindole (DAPI). Slides were visualized using a fluorescence microscope LSM 800 (Zeiss, Jena, Germany).

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).