Fig. 2. Palmitoylation attenuates the autophagic degradation of NOD2.

a Immunoblot analysis showing NOD2 protein level in HCT116 cells treated with proteasome inhibitors MG132 (10 μM), carfilzomib (100 nM), or autolysosome inhibitor ammonium chloride (NH₄Cl, 20 mM), bafilomycin A1 (Baf A1, 0.2 μM), or chloroquine (CQ, 50 μM) for 6 h. b Quantification of relative protein level of NOD2 in (a). c Immunoblot analysis showing NOD2 degradation in HCT116 cells treated with DMSO or 2BP (100 μM, 24 h) upon starvation-induced autophagy activation under Earle’s balanced salt solution (EBSS)-cultured condition for 0, 3, 6 or 9 h. d Quantification of relative protein level of NOD2 in (c). e, h Immunoblot analysis showing protein level of Flag-NOD2 in wild-type (WT) and BECN1 (e) or ATG5 (g) knockout (KO) HEK293T cells. Cells were transfected with Flag-NOD2 for 24 h, and then treated with CHX (100 μg/ml) for 0, 3, 6 and 9 h. f, h Quantification of relative NOD2 remaining in (e and g). i–l Immunoblot analysis showing protein level of Flag-NOD2 in WT and BECN1 (i, j) or ATG5 (k, l) KO HEK293T cells treated with DMSO or 2BP (100 μM, 24 h). j, l Quantification of relative protein level of NOD2 in (i and k). In b, d, f, j, and l, 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–e, g, i, k, similar results are obtained from three independent biological experiments.