Fig. 2: NAGK is required for MDP recognition.
From: Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation
a, NAGK expression in wild-type and NAGK-knockout KBM-7-IL-1BmScarlet cells by immunoblot (representative of three independent experiments). b, Wild-type and NAGK-knockout KBM-7-IL-1BmScarlet cells were treated with the indicated doses of L18-MDP (left), MDP (middle) or C12-ie-DAP (right) for 16 h. Data are mean ± s.e.m of n = 3 independent biological samples. Two-way ANOVA with Šídák’s multiple-comparisons test. c, NAGK expression in wild-type and NAGK-knockout NOD2-expressing HEK cells (representative of two independent experiments). d,e, Wild-type and NAGK-knockout NOD2-expressing HEK cells were stimulated as indicated and IL-8 production was determined by enzyme-linked immunosorbent assay (ELISA) after 16 h. Data are mean ± s.e.m of n = 3 independent biological samples. Two-way ANOVA with Šídák’s multiple-comparisons test. f, RIPK2 ubiquitination and NF-κB and MAPK activation markers following L18-MDP stimulation in wild-type or NAGK-knockout THP-1 cells. Ubiquitinated proteins were enriched by pulldown of endogenous ubiquitinated protein bound to an immobilized ubiquitin-associated domain (UbA). Blot represents one of three biological replicates. g, Heat map of significantly varying phosphosites in wild-type or NAGK-knockout THP-1 cells treated with L18-MDP for 1 h (Student’s t-test FDR < 0.05, z-scores). Fisher’s exact t-test of significantly upregulated phosphosites upon L18-MDP treatment (P < 0.02; enrichment terms include Gene Ontology Biological Process, KEGG and Gene Ontology Molecular Function). h, Schematic representation of the NOD2–NanoBiT luminescence assay. i, Immunoblot of NAGK expression in wild-type and NAGK-knockout HEK 239T cells (representative of two independent experiments). Cells were transfected with the constructs shown in h and treated with L18-MDP for 24 h. NOD2 dimerization was measured as the luminescence induced by NOD2–NOD2 interaction, normalized with the positive and negative controls. Data are mean ± s.e.m of n = 3 independent biological samples. Two-way ANOVA with Šídák’s multiple-comparisons test.
