Anti tubulin rabbit polyantibody

Tissues were minced in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH7.5, 0.5% NP-40) supplemented with protease inhibitor, homogenized using a mini-homogenizer, and gently rotated for 30 min at 4 ºC. The obtained tissue lysates were centrifuged at 18,000 ×g at 4 ºC for 20 min, and the supernatant was collected and measured for protein concentration. Tissue lysates that contained equal amounts of proteins were loaded on the NuPAGE Bis-Tris protein gel (Invitrogen) for immunoblot analysis 19 (link). Primary antibodies used were anti-NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000); anti-ASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000); anti-Casp1 (p10) mouse antibody (Adipogen, AG20B0044C100, 1:1000); anti-tubulin rabbit polyantibody (Santa Cruz, Dallas, TX, USA, SC-5286, 1:200); anti-Nek7 rabbit antibody (Abcam, Cambridge, MA, USA, ab133514, 1:10000); and anti-IL-1β goat antibody (R&D systems, Minneapolis, MN, USA, AF401NA, 1:2000).

Protein lysates from cells or liver tissues were separated on a 4%–12% NuPAGE Bis‐Tris protein gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Chicago, IL, USA). Blots were blocked with 5% nonfat milk (Nestle, Jacksonville, IL, USA), then incubated with primary antibodies in Tris‐buffered saline containing 0.1% Tween 20 (Sigma) and 5% nonfat milk, followed by HRP‐conjugated anti‐rabbit antibody (Cell Signaling, Danvers, MA, USA, 7074S, at 1:3000 dilution) or anti‐mouse antibodies (Cell Signaling, 7076S, at 1:2500 dilution). Primary antibodies used in this study were anti‐NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000); anti‐ASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000); anti‐Casp1 (p10) mouse antibody (Adipogen, AG20B0044C100, 1:1000); anti‐tubulin rabbit polyantibody (Santa Cruz, Dallas, TX, USA, SC‐5286, 1:200); anti‐Nek7 rabbit antibody (Abcam, Cambridge, MA, USA, ab133514, 1:10000); and anti‐IL‐1β goat antibody (R&D systems, Minneapolis, MN, USA, AF401NA, 1:2000).

Immunoblot analysis was conducted as described [20 (link)]. Primary antibodies used were: Anti-IL-1β goat antibody (R&D systems, Minneapolis, MN, USA, AF401NA, 1:2000); anti-tubulin rabbit polyantibody (Santa Cruz Biotechnology, Dallas, TX, USA, SC-5286, 1:200); anti-Casp1 (p10) mouse antibody (Adipogen, San Diego, CA, USA, AG20B0044C100, 1:1000); anti-ASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000); anti-NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000); and anti-Nek7 rabbit antibody (Abcam, Cambridge, MA, USA, ab133514, 1:10000). RNA-bee (Tel-Test, Friendswood, TX, USA) was used to extract mRNAs from livers, which were then subjected to qPCR analysis [20 (link)]. The housekeeping genes, including the Hprt and Rplp0 genes, were used as reference genes for normalization.