Anti mlkl phospho s345 antibody

Immunofluorescence was performed on frozen liver sections, as previously described^{38 (link)}. Liver sections were stained with the following primary antibodies: FITC Mouse Anti-iNOS/NOS Type II (1:200; BD Transduction Laboratories™, San Jose, CA, USA), PE anti-mouse CD206 (MMR) (1:200; BioLegend Inc., San Diego, CA, USA), Anti-actin, α-Smooth Muscle antibody (1:300; Sigma, St Louis, MO, USA), Anti-mouse MLKL (1:400; Biorbyt, San Francisco, CA, USA), Anti-MLKL (phospho S345) antibody (1:400; Abcam), Anti-RIPK3 (1:400; Abcam), RIP(D94C12) XP Rabbit mAb (1:400; Cell Signaling Technology, MA, USA), anti-NLRP3 antibody (1:300; Abcam), anti-ASC antibody (1:200, Cell Signaling Technology), and anti-cleaved caspase-1 (1:200; Cell Signaling Technology). For indirect immunofluorescent staining, liver sections were incubated with the following secondary antibodies: PE-conjugated donkey anti-rabbit IgG for p-MLKL, MLKL, RIPK3, and SMA (1:500); Alexa Fluor 488-conjugated donkey anti-rabbit IgG for p-MLKL, MLKL, RIPK3, RIPK1, NLRP3, ASC, and cleaved caspase-1 (1:500). Nikon Inverted Fluorescence Microscope ECLIPSE Ti and NIS-Elements F 3.0 Software (Nikon Corporation, Tokyo, Japan) were applied for image capture.

A suitable amount of lung tissue stored in -80°C freezer was homogenated and lysed in RIPA buffer containing proteinase and phosphatase inhibitor cocktail (CWBIO Co. Ltd., China). After protein concentrations were assessed using the BCA protein assay kit (CWBIO Co. Ltd., China), protein samples were boiled in loading buffer for 10 min. 40 μg protein samples were then run on a 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA) at 250mA for 2h. The membrane was blocked with 5% fat-free milk in TBST (0.1% Tween-20 in TBS) for 2h at room temperature and incubated with primary antibodies at 4°C over night. Primary antibodies used in this study were anti-cIAP2 antibody(1:1000, R&D system), anti-RIPK1 antibody (1:1000, Novus), anti-RIPK3 antibody (1:1000, Abcam), anti-MLKL (1:1000, Abcam), anti-RIP3 (phospho S227) antibody (1:2000, Abcam), anti-MLKL (phospho S345) antibody (1:1000, Abcam), and anti-GAPDH antibody(1:4000, CWBIO Co. Ltd). Then the membranes were washed 3 times in TBST and incubated with goat-anti-mouse/rabbit antibody for 2h at room temperature. After being washed 3 times in TBST again, the protein bands were visualized by fluorography using ECL (enhanced chemiluminescence) reagents (Millipore, USA) and analyzed by image J.