Fig. 1: Phosphoproteomic analysis and use of targeted inhibitors in human MoDCs reveals a role for endocytosis in synergistic enhancement of cytokine production in newborns.

Newborn and adult MoDCs were stimulated with 50 µM R848, 100 ug/ml TDB, combination, or vehicle control for 30 min. a, b Pathway overrepresentation analysis (hypergeometric test with Benjamini Hochberg correction) of phosphopeptides quantified through mass spectrometry reveals synergistic enrichment of both cytokine- and endocytosis pathways by R848+TDB in newborn, but not adult, cells (hypergeometric test). Significant increase in phosphorylation (paired Student’s t test) in newborn MoDCs of PRKCD at Ser645 and its substrate, STAT1 at Ser727, (c, d) are shown using box-whisker plots (center:mean, boxes:75th and 25th percentile, whiskers: minimum and maximum values) was confirmed by western blotting (e), demonstrating prolonged phosphorylation of these two residues after stimulation with R848+TDB. f–h Inhibition of endocytosis with a clathrin inhibitor reduced PRKCD phosphorylation and subsequent TNF-α production by neonatal MoDCs. n = 9 newborns, 4 adults, error bars indicate mean+SEM. Statistical comparisons in (c, d, f, and h) are indicated by connecting lines and employed two-tailed Wilcoxon rank-sum test (*p < 0.05, **p < 0.01, ***P < 0.001). Source data are provided as a Source Data file.