Caspase 1 sc 514

Antibodies recognized NLRP3 (Cryo-2; Adipogene), FLAG (M2; Sigma-Aldrich), ASC (8E4.1; Genentech), caspase-1 (sc-514; Santa Cruz Biotechnology, Inc.), IL-1β (GTX74034; GeneTex), phospho-NLRC4 S533 (GEN-82 clone 3–3; Genentech), and NLRC4 (hamster monoclonal 1D4 for immunoprecipitation and a rabbit polyclonal for blotting; Genentech). Other reagents included Z-VAD-FMK (MBL), S. typhimurium flagellin (InvivoGen), LPS from Escherichia Coli O111:B4 (Invivogen), Pam3CSK4 (Invivogen), and DOTAP transfection reagent (Roche). LDH release was measured with a Cytotoxicity Detection kit^plus (Roche). Secreted IL-1β and TNF were measured with Meso Scale Discovery mouse kits.

The total brain^{66 (link)} and microvessel protein extracts^{64 (link)} from mouse homogenized cerebral cortex were prepared for western blotting. Samples containing equal amounts of protein were loaded onto 10%-13.5% acrylamide denaturing gels (SDS-PAGE) for protein separation. Proteins were then transferred to an immobilon polyvinylidene difluoride membrane for 1 h at 50 V. Then membrane were blocked in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.1% Tween 20 (TBS-T) containing 5% fat-free milk powder for 1 h and probed overnight at 4 °C with antibodies against zonula occludens-1 (ZO-1) (Invitrogen, Carlsbad, CA, USA), spectrin and MMP-9 (Millipore, Billerica, MA, USA), P2RX₇ (Abcam, Cambridge, MA, USA), FADD (Epitomics, CA, USA) and β-actin (Sigma Chemical, St. Louis, MO, USA). Caspase-1 (sc-56036, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to detect ProCaspase-1, Caspase-1 (sc-514, Santa Cruz Biotechnology) was used to detect Caspase-1(p10), IL-1β (sc-7884, Santa Cruz Biotechnology) was used to detect ProIL-1β, and IL-1β (D4T2D, Cell Signaling Technology, Danvers, MA, USA) was used to detect IL-1β (p17). Bands were visualized by enhanced chemiluminescence (Amersham Life Science, Buckinghamshire, UK). Immunoreactive band intensities were analyzed by Image J software (NIH). The band was normalized against the corresponding β-actin band used as loading control.