SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes

Shi, Chong-Shan; Nabar, Neel R.; Huang, Ning-Na; Kehrl, John H.

All lysates were mixed with 4x NuPage LDS Sample Buffer and heated at 100°C for 10 min, followed by separation on a 4-20% Tris-Gylcine Gel (Invitrogen) and transfer to a nitrocellulose membrane using the iBlot Gel Transfer System (Invitrogen). The membrane was blocked with 5% nonfat milk (or 5% BSA) in TBST (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h and incubated at 4°C overnight with the primary Ab in TBST with 5% BSA. The appropriate secondary Abs conjugated to HRP were used to detect the protein of interest via ECL. When necessary, membranes were stripped using Restore Plus Western Blot Stripping Buffer (Thermo, 46430) following the manufacturers protocol, re-blocked, and reblotted. Images were acquired either by exposure on film (Amersham Hyperfilm ECL) or using the iBright 1000FL (Invitrogen). Blots were scanned and imported into Photoshop as unmodified tagged image files; quantification of band intensity was performed using standard methods on ImageJ (NIH) and presented relative to the appropriate housekeeping gene.