Fig. 4

The FCAS2-causing mutation results in NLRP12 haploinsufficiency that impairs MDP tolerance. a The FCAS2-causing NLRP12 mutant shows impaired NOD2 suppression activity. HEK293T-based NF-κB-Luciferase Assay as described in Fig. 2c. Depicted are mean ± SEM (n = 3). b NLRP12 interacts with the chaperone protein HSP90. HEK293T cells were transfected with Myc-tagged NLRP12 constructs for 48 h. Myc-tagged NLRP12 was precipitated from lysates and investigated for interaction with HSP90 using western blot analysis. c The R284X mutant failed to interact with the chaperone protein HSP90. HEK293T cells were transfected with Myc-tagged NLRP12 constructs for 48 h. Myc-tagged NLRP12 was precipitated from lysates and investigated for interaction with HSP90 using western blot analysis. d Representative co-immunoprecipitation experiment with the indicated plasmids. NLRP12-specific immunoprecipitation was performed on native protein lysates by western blot analysis using anti-FLAG (M2) antibody. Immunoprecipitation by using antibodies against FLAG (M2). NLRP12 and RIPK2 complexes with NOD2 were detected by immunoblot (IB) using anti-NLRP12 antibody. Non-precipitated protein lysates were used as input controls. The symbol * refers to non-specific band. e MDP-induced secretion of TNF-alpha by THP-1 parental (wild-type), THP-1 NLRP12^−/− and THP-1 NOD2^−/− cell lines by ELISA. The cells were incubated for either 6 h, 12 h, 24 h or 48 h with MDP at 10 μg/mL before being spun down for collecting the supernatant. All experiments were performed in triplicate. f ELISA analysis of TNF-alpha secretion by PBMCs of healthy donors and the twin patients carrying the nonsense R284X mutation in the NLRP12 gene. g Sequencing electropherograms of NLRP12 cDNA are depicted before and after treatment of patient’s PBMCs by 30 μg/mL of cycloheximide (CHX) for blocking nonsense-mediated mRNA decay