Nb600 501h

Cell lysates, serum samples and spleen homogenates denatured in loading buffer containing SDS and 100 mM DTT were boiled for 10 min before SDS–PAGE-separated proteins were transferred to PVDF membranes. Immunoblotting with primary antibodies against caspase-1 (1/3,000, kind gift of Dr Peter Vandenabeele, Ghent University), ASC (1/1,000, AG-25B-0006, Adipogen), IL-1β (1/2,000, GTX74034, GeneTex), caspase-11 (1/1,000, NB120-10454, Novus Biologicals), Ub-HRP (1/1,000, sc-8017, Santa Cruz), HMGB1 (1/100, 18256, Abcam), and β-actin (1/20,000, NB600-501H, Novus Biologicals) was followed by secondary anti-rabbit and anti-rat HRP antibodies (1/5,000, 111-035-144 and 112-035-143, Jackson Immunoresearch). Ponceau S Solution was purchased from Sigma-Aldrich.

Two retinas from an P8-10 animal were dissected in cold PBS and homogenized in a 2 mL tube with 0.3 mL lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris pH 8.0, 1 mmol/L CaCl2, 1% NP40, 0.1% N-dodecyl-β-D-maltoside, 3 mmol/L MgCl₂, and DNAseI 80 units/mL (Roche), 1× complete ultra EDTA-free protease inhibitor cocktail [Roche]) using an electrical homogenizer (Omni). Proteins were extracted for 40 minutes on ice with gentle shaking. Cell debris and unsolubilized material were removed by 20 000×g centrifugation at 4°C for 30 minutes. One hundred four microliter supernatant, 40 µL 4× LDS (Nupage), and 16 µL 1 mol/L dithiothreitol were combined and incubated at 70°C for 10 minutes. Forty microliters of the sample were loaded per lane. VE-cadherin was detected using Millipore anti-VE-cadherin clone Vli37 (MABT886) at 1:1000. PECAM (cell signaling, 77699) and β-actin (Novus, NB600-501H) were probed as loading control.