Fig 5. Human NLRP1 does not require a Walker A motif for inflammasome assembly.

(A) 293T cells were transfected with 100ng of CMSCV empty vector (V) or plasmids encoding mouse wild type HA-NLRP1B¹²⁹ (WT) or mutant HA-NLRP1B¹²⁹ either containing the K137R mutation in the Walker A (WA) ATP-binding site or the constitutively active ΔLRR mutant. The cells were co-transfected with vectors encoding mouse CASP1 and IL-1β. Cells expressing mouse NLRP1B were treated with LeTx for 16h where indicated. Non-boiled lysates were subject to immunoblot (IB) with anti-HA antibody, whereas boiled lysates were subject to immunoblot with anti-IL-1β to detect CASP1-dependent processing of IL-1β into the mature p17 fragment. (B) 239T cells were transfected with 100ng empty vector (V) or plasmids encoding hNLRP1-MYC wild-type (WT), Walker A mutant 1 K340A (WA1), Walker A mutant 2 K340R (WA2) or ΔLRR expression plasmids. The transfection complexes also contained 200ng mCASP, 200ng mIL-1β and 1ng hASC expression vectors. For both A and B, data shown are representative of at least three similar experiments.