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.

For standard immunoblotting, cells were lysed in Buffer A containing 20 mM HEPES (pH 7.4), 50 mM β-glycerophosphate, 1% (v/v) Triton X-100, 2 mM EGTA, and cOmplete protease inhibitor cocktail (Sigma, 11836170001) plus PhosStop (Sigma, 04906837001) phosphatase inhibitor tablets for 30 min. Lysates were cleared by centrifugation at 14,000 rpm for 10 min and the supernatant collected. For separation of Triton soluble and insoluble fractions, cells were lysed in Buffer A and centrifuged as before. The supernatant was considered the Triton soluble fraction, while the pellet was directly mixed with 1x NuPage LDS Sample Buffer (Invitrogen, NP0008) and heated at 100°C for 30 min (Triton insoluble fraction). For nuclear fractionation, cells were lysed with Buffer B, which is Buffer A substituting 0.5 (v/v) NP-40 for Triton X-100. After centrifugation, the supernatant was considered the cytosolic fraction. The resultant pellet was washed 4-6 times with Buffer B and then lysed in Buffer B + 0.5% (v/v) SDS for 30 min, this was considered the nuclear fraction. Inflammasome activation was measured by immunoblotting cell culture supernatants for mature IL-1β and cleaved caspase-1 as previously described 49 . For immunoprecipitations, cells were lysed with Buffer C, which is Buffer A + 0.5% (w/v) CHAPS and incubated with either GFP or FLAG beads for 3 h. Immunoprecipitates were washed 4-6 times and resuspended in 1x NuPage LDS buffer to release captured proteins prior to immunoblotting.