Odessey blocking buffer

Cell monolayers were washed with 1X PBS and lysed in 1X RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with protease and phosphatase inhibitors (1 tablet per 10 ml; Pierce). DNA was degraded using 250 unitsbenzonase (EMD Milipore). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at RT in Odessey blocking buffer (Licor) or 4% BSA and incubated overnight at 4°C with the following antibodies: pNF-κBSer536, 1:1000 (Cell Signaling 3033S), STAT1 1:1000 (Cell Signaling 9172S), pSTAT1 Tyr7011:1000 (Cell Signaling 9177S); IRF3 1:1000 (Cell Signaling 4302), pIRF3 Ser396 1:1000 (Cell Signaling 4947); Beta Actin 1:5000 (Abcam 6276), Tubulin 1:5000 (Abcam 179513). Membranes were washed 3x in 1X TBS 0.1% Tween 20 andincubated with appropriate secondary antibody (Licor) for 2 h at RT (20°C) prior to imaging on Odessey Fc Dual-Mode Imaging System (Licor).

1.5X10⁶ cells per well were plated for western blotting experiments. After mentioned time point, cells were washed with ice cold PBS before their incubation with Buffer A solution (20mM HEPES, 10mM NaCl, 1.5mM MgCl₂, 0.2mM EDTA and 0.5%v/v Trition-X-100) with 1X Protease Arrest (G-Biosciences) for 15 minutes on ice for lysis. Cell lysate was centrifuged at 4°C at 6000g for 10 minutes and supernatant was collected. Protein quantification was done using BSA as standard in Bradford’s assay. Protein sample was mixed with 6X loading dye and subjected to SDS PAGE and transferred to nitrocellulose membrane for immunoblotting. Blocking was done for an hour with Odessey blocking buffer (LI-COR Biosciences) in 1:1 dilution with 1X PBS at room temperature. Blots were immunoblotted with primary and then with secondary antibody made in blocking buffer. Blots were imaged with Odessey Infra Red Imaging system(LI-COR Biosciences).

Protein samples were harvested after lysing cells in radioimmunoprecipitation buffer (10 mM phosphate pH 7.4, 137 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) supplemented with protease inhibitor cocktail (Sigma; St. Louis, MO) and phosphatase inhibitors cocktail II and III (Sigma; St. Louis, MO). Protein extracts were resolved in 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Millipore, Billerica, MA). Blots were blocked in Odessey blocking buffer (LI-COR Biosciences, Lincoln, Nebraska). Western blotting detection was done after incubation with primary antibodies (anti-p-MLKL antibody, Cat#ab196436, Abcam; anti-total-MLKL, Cat#AP14272B, Abcepta; anti-cleaved caspase 3 antibody, Cat#9661, Cell Signaling Technology; and anti-beta-actin antibody, Cat#4967, Cell Signaling Technology) infrared dye-conjugated secondary antibody (Donkey-anti-Rabbit IgG-800CW conjugated, Cat#92632213, LI-COR Biosciences) using Odssey Scanner (LI-COR Biosciences, Lincoln, Nebraska).