Anti mouse antibodies

BW25113(ycbB, relA’) was grown in the −CRO and +CRO conditions in BHI agar plates as performed for the preparation of sacculi. Bacteria were lysed with 4% SDS at 96 °C for 45 min. Crude bacterial extracts were separated by SDS-PAGE. Lpp and RpoA (used as a loading control) were detected with polyclonal rabbit and mouse antibodies provided by J.F. Collet and C. Beloin, respectively. Primary antibodies were diluted at 1/10,000 (v/v). Detection was performed with peroxidase-coupled anti-rabbit (Sigma) and anti-mouse antibodies (Sigma) according to the manufacturer instructions of the Pierce^TM ECL Western Blotting Substrate (Thermo Scientific). Secondary antibodies were diluted at 1/2000 (v/v).

The FDB or soleus muscles from mice were homogenized by sonication in cold RIPA lysis buffer, containing 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM BAPTA, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, and Protease Inhibitor Cocktail (Roche Diagnostics, Germany), as described previously [20 (link)]. The samples were loaded onto a 12% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The primary antibodies used were: anti-NLRP3 (1:500) from R&D Systems (MAB7578, Minneapolis, MN, USA), anti-IL-1β (1:500; SC-32294), anti-caspase-1 (1:500; SC-56036), anti-ASC (1:1000; SC514414), and anti-GSDMD (1:500; SC-393581) from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-GADPH (1:20000) from Sigma-Aldrich (G9545; St. Louis, MO, USA). After washing, the membranes were incubated for 1.5 h with secondary anti-rabbit, anti-rat, or anti-mouse antibodies, as appropriate (Sigma-Aldrich, St. Louis, MO, USA). Western blotting detection reagents (LumiFlash™ Infinity Chemiluminescent Substrate, Taipei, Taiwan) were used following the manufacturer’s instructions, and chemiluminescence was detected using a ChemiDoc Imaging System from Bio-Rad (Hercules, CA, USA). The intensity of the bands was quantified by densitometry, with the use of ImageJ software version 1.44p (NIH, Bethesda, MD, USA).

Protein samples were separated by 12% SDS PAGE and analyzed for the presence of GST- or His₆-tag by WB with rabbit anti-GST (Sigma) and mouse anti-His₆ antibodies (Sigma), respectively. LEDGF/p75 was detected by mouse anti-LEDGF monoclonal antibody (Santa Cruz Biotechnology). For the detection of IN_HA, an anti-integrate rabbit serum was used (a kind gift from Dr. M. Isaguliants). For the detection of Ku70_3FLAG, an anti-FLAG M2 HRP-conjugated antibody (Sigma) or anti-Ku70 rabbit antibody (Sigma) were used. HRP-conjugated anti-rabbit (Sigma) and anti-mouse antibodies (Sigma) were used as secondary antibodies. Visualization of specific protein bands was performed with Clarity Western ECL substrate (Bio-Rad) on ChemiDoc MP system (Bio-Rad).

Protein samples were separated by 12% SDS PAGE and analyzed for the presence of GST- or His₆-tag by WB with rabbit anti-GST (Sigma) and mouse anti-His6 antibodies (Sigma), respectively. For the detection of IN_HA, an anti-HA monoclonal antibody (Invitrogen) was used. For the detection of Ku70_3FLAG, an anti-FLAG M2 HRP-conjugated antibody (Sigma) or anti-Ku70 rabbit antibody (Sigma) were used. Mouse anti-human tubulin clone 12G10 mAb (Developmental Studies Hybridoma Bank at the University of Iowa) as primary antibodies was used. HRP-conjugated anti-rabbit (Sigma) and anti-mouse antibodies (Sigma) were used as secondary antibodies. Visualization of specific protein bands was performed with Clarity Western ECL substrate (Bio-Rad) on ChemiDoc MP system (Bio-Rad).