Irdye 800cw secondary antibody

Expression of translational mCherry fusions was induced in WT V. cholerae with 0.2% arabinose for pBAD33 or 1 mM IPTG for pHL100 and lacZ::P_tac in LB and grown to OD₆₀₀~0.6. Cells were harvested by centrifugation (9500 g, 15 min) at room temperature and resuspended in 1% SDS + 10 mM dithiothreitol (DTT) lysis buffer. Resuspended cells were incubated at 95°C for 3 min, then sonicated 4 × 5 seconds at 20% amplitude. Standard Western blots against mCherry were performed using polyclonal mCherry antibody (Genetex #GTX59788) and detection by IRDye 800CW secondary antibody (Li-cor #926–32211). After imaging for mCherry, the same blots were then re-incubated with monoclonal RpoA antibody (BioLegend # 663104) detected by IRDye 800CW secondary antibody on an Odyssey CLx imaging device (Li-cor).

Whole spinal cord from Ipsilateral T13/L1 was sonicated in a mixture containing extraction buffer (Invitrogen) and protease inhibitors (Sigma). Ice-cold tissue samples were centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was removed, and the protein concentration for each sample was quantified using the Bradford method. Samples were heated to 75°C for 10 min and loaded into a standard polyacrylamide Bis-Tris gel (Invitrogen). SDS-PAGE was performed in 3-(N-morpholino)-propanesulfonic acid running buffer (Invitrogen) at 175 V for 1.25 h. Protein was transferred onto a nitrocellulose membrane using the iblot dry transfer system (Invitrogen). The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences) for 1 h and incubated with a primary antibody in blocking buffer overnight at 4°C. The following day, the membrane was washed in 1× PBS containing Tween 20 (0.1%) and then incubated in blocking buffer containing either goat anti-rabbit (NLRP3) or goat anti-mouse (B-actin) (LI-COR) IRDye 800CW secondary antibody at a concentration of 1:10,000 (LI-COR) for 1 h at room temperature. Protein expression was quantified using an Odyssey Infrared Imager (LI-COR) and expressed as a ratio to their housekeeping protein. Primary antibodies included rabbit anti-rat NLRP3 monoclonal antibody (1:1000; Abcam), and mouse anti-rat β-actin (1:200,000; Sigma-Aldrich).