Irdye 800 labeled anti rabbit igg 611 132 002

The cultured supernatants and cell lysates were collected at the indicated time points after Stx2 treatment, and protein in the cell-free supernatants was concentrated using the methanol-chloroform precipitation method (27 (link)). The cell pellets were lysed with the RIPA Lysis buffer (89901, Thermo) supplemented with a 1:50 diluted protease inhibitor cocktail tablet (EDTA-free protease inhibitor cocktail tablet, Roche). Such prepared samples were then mixed with the equal volume of 2× SDS-loading buffer and detected for pro-IL-1β/IL-1β and pro-caspase-1/caspase-1 by immunoblotting. β-Actin was used as the positive control. Immobilized proteins were incubated with primary antibodies against IL-1β (sc-52012; 1:1,000), caspase-1 (AG-20B-0042; 1:1,000), and β-actin (4967S; 1:1,000), and followed by incubation with the secondary antibodies (IRDye 800-labeled anti-rabbit IgG; 611-132-002; 1:10,000 (Santa Cruz Biotechnology). The protein levels were detected using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE).

The cultured supernatants and bacterial lysates were collected at the indicated time after treatment (Liu et al., 2015 (link)). Proteins in the supernatants were extracted using the methanol – chloroform approach, and the cell pellets were lysed with the RIPA Lysis buffer (89901, Thermo) supplemented with a protease inhibitor cocktail (Roche). Finally, all the protein pellets were mixed with the SDS loading buffer. Both of the obtained proteins were applied to detection for pro-IL-1β/IL-1β, and pro-Caspase-1/Caspase-1 by immunoblotting. β-actin was chosen as the positive control. The antibodies against IL-1β (sc-52012), Caspase-1 (sc-56036), β-actin (4967S), and the secondary antibodies (IRDye 800-labeled anti-rabbit IgG; 611-132-002) were obtained from Santa Cruz Biotechnology. The proteins were quantified using an Odyssey infrared imaging system (LI-COR, Lincoln, NE).