Fig. 5. R444C variant promotes NOD2s palmitoylation through enhancing its binding ability with ZDHHHC5.

a Immunoblot analysis showing degradation of NOD2s-WT/R444C or NOD2l-WT/R471C, under CHX (100 μg/ml) treatment for 0, 3, 6, and 9 h. b Quantification of NOD2s-WT/R444C or NOD2l-WT/R471C in (a). c Streptavidin blot detection of palmitoylated NOD2s-WT/R444C or NOD2l-WT/R471C in HEK293T cells transfected with Flag-tagged NOD2s-WT/R444C or NOD2l-WT/R471C by acyl-biotin exchange (ABE) assay with or without hydroxylamine (HAM, 1 M). The S-palmitoylated NOD2 in immunoprecipitated samples were detected using anti-Flag antibody. d Immunofluorescence staining of Flag-NOD2s-WT or Flag-NOD2s-R444C in HEK293T cells transfected with the same amount of Flag-NOD2s-WT or Flag-NOD2s-R444C plasmids, with or without 2-BP (100 μM, 24 h) treatment. The nucleus is stained blue with DAPI. Scale bars, 10 μm. e Quantification of the NOD2 intensity (30 cells per sample). r.f.u. relative fluorescence unit. f, g Co-immunoprecipitation and immunoblot analysis showing the interaction of NOD2 and ZDHHC5 (f) or SQSTM1 (g) in HEK293T cells transfected with Flag-tagged NOD2s-WT/R444C or NOD2l-WT/R471C and HA-ZDHHC5 (f) or HA-SQSTM1 (g) for 24 h. h Immunoblot analysis showing the interaction of NOD2 and ZDHHC5 or SQSTM1 in MDP (10 μg/ml)-induced NOD2s-WT/R444C THP-1-DM cells. Cells were differentiated with PMA (100 ng/ml) overnight and subsequently treated with Dox for 10 h. In a–c and f–h, we adjusted the amount of transfected plasmids or Dox to make the protein level of NOD2 the same. In b and e, all error bars, mean values ± SEM, P values were determined by unpaired two-tailed Student’s t-test of n = 3 independent biological experiments. *P < 0.05; **P < 0.01; ns not significant. For a, c, d, and f–h, similar results are obtained from three independent biological experiments.