Horseradish peroxidase conjugated secondary antibody

Ly6G⁺ neutrophils, isolated by flow cytometry, were homogenized in extraction buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1 % Triton X-100, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 2 mM Na pyrophosphate, 10 mM Na β-glycerophosphate, 1 mM Na orthovanadate, 1 mM phenylmethanesulfonylfluoride, 1× protease and phosphatase inhibitor cocktail [Sigma]). The protein samples (50 μg) were resolved in a 12 % SDS-PAGE Mini-Protean Precast gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (PerkinElmer). The membrane was blocked in PBS containing 0.1 % Tween 20 and 7 % non-fat milk, and then incubated at 4 °C overnight with an antibody against IL-36γ (1:200, Santa Cruz, sc-168163) or β-actin (1:50000, Abcam, mab150), followed by 1 h at room temperature in the appropriate secondary horseradish peroxidase-conjugated antibody (Cell Signaling Technology). The antibodies were detected using the Western Lightning Plus-ECL chemiluminescence substrate (Perkin Elmer).

DNA was extracted and quantified as mentioned before. One thousand nanograms of DNA were diluted in TE buffer. Subsequently, DNA was denatured in 0.1 M of NaOH at 95 °C for 10 min and single chains were stabilized in 1 M of ammonium acetate. DNA was pipetted into nitrocellulose membranes, which were allowed to dry for 30 min at 37 °C. Membranes were exposed to UV light for 1 min to produce crosslinks between DNA and membranes. Then, membranes were blocked with 5% dry milk in TBS with 0.1% Tween and incubated with primary antibody against 5mC (Millipore, 1:1000) at 4 °C, overnight. On the next day, membranes were incubated with secondary horseradish peroxidase conjugated antibody (Cell Signaling Technology) anti-mouse (1:5000), for 1 h at room temperature. As for western blot, the blots were detected using enhanced chemiluminescence and quantified using ImageJ software. Sybergreen I nucleic acid stain (Molecular Probes, 57567, Invitrogen, Carlsbad, CA, USA) was used as loading control.

Whole cell extracts were lysed on ice in RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.4, 1% NP-40, 0.1% SDS), supplemented with complete EDTA-free protease inhibitor cocktail (Roche). Total protein concentrations were quantified using the BCA protein kit (Life Technologies), and cell lysates containing 20 μg of protein were subjected to electrophoretic separation on denaturing polyacrylamide gels under reducing conditions, followed by transfer to PVDF membranes. The latter were then probed with a mouse anti-myc antibody (1:1000, Cell Signalling Technologies), a rat anti-MLKL antibody (1:2000) [15 (link)] and a rabbit anti-GAPDH antibody (1:2500, Trevigen), followed by the appropriate secondary horseradish peroxidase-conjugated antibody (1:3000, Cell Signalling Technologies). The signal was visualized using the chemiluminescent ECL reagent (Life Technologies).