Skip to main content
. Author manuscript; available in PMC: 2020 Apr 5.
Published in final edited form as: Science. 2019 Mar 14;364(6435):82–85. doi: 10.1126/science.aau1208

Fig. 2. LT and VbP induce proteasome-mediated degradation of NLRP1B.

Fig. 2.

(A) Diagram of NLRP1B. Single-letter abbreviations for the amino acid residues are as follows: F, Phe; K, Lys; L, Leu; M, Met; R, Arg; and S, Ser. (B and C) HEK 293T cells stably expressing mCasp1 (“m” denotes mouse) were transiently transfected with the indicated constructs (2 μg) for 24 hours, before cell viability was evaluated by lactate dehydrogenase (LDH) release (B) and expression was evaluated by immunoblotting (C). Residues that were mutated to create start sites are indicated. Data are means ± SEM of three biological replicates. ***P < 0.001 and **P < 0.01 by two-sided Student’s t test compared with mock. GST, glutathione S-transferase; GAPDH, glyceraldehyde phosphate dehydrogenase. (D) HEK 293T cells stably expressing mCasp1 were transiently transfected with a construct encoding GST-NLRP1B (30 ng). After 24 hours, cycloheximide (CHX, 100 mg/ml; used to block new protein synthesis), LT (1 μg/ml), and VbP (10 μM) were added to the indicated samples, which were then incubated for an additional 6 hours. FL, full-length. Asterisks indicate background bands. (E) HEK 293Tcells stably expressing mCasp1 were transiently transfected with a construct encoding V5-GFP-NLRP1B-FLAG (0.1 μg). After 24 hours, cells were treated with dimethyl sulfoxide (DMSO), bortezomib (Bort, 20 μM), MG-132 (20 μM), or Me-Bs (20 μM) for 30 min before the addition of either LT (1 μg/ml, 6 hours) or VbP (10 μM, 6 hours). Protein levels were evaluated by immunoblotting. (F) RAW 264.7 cells were treated with DMSO, bortezomib (20 μM), or MG-132 (20 μM) for 30 min before the addition of LT (1 μg/ml, 3 hours) or VbP (2 μM, 6 hours). Protein levels were evaluated by immune-blotting. Data are representative of three or more independent experiments.