Activation of neddylation in T cells upon TCR ligation and blood-stage <i>P</i>. <i>yoelii</i> 17XNL infection.

(A) Immunoblotting of total cell extracts from splenic T cells of C57BL/6 mice stimulated with CD3/CD28 Dynabeads for various times (different parts of the same gel were grouped together with a dividing line). Uba3 and NEDD8-conjugated proteins were detected using anti-Uba3 and anti-NEDD8 antibodies, respectively. (B) Splenic T cells purified from C57BL/6 mice were incubated with MLN4924 (0.1, 0.5 μM) or DMSO (–) for 1h, then left untreated or stimulated with CD3/CD28 Dynabeads for 48h, NEDD8-conjugated cullins were detected by probing with anti-NEDD8 antibody. (C) Immunoblotting for Uba3 and NEDD8-conjugated proteins in splenic T cells isolated from C57BL/6 mice at indicated time points of blood-stage P. yoelii 17XNL infection, Uba3 and neddylated proteins were detected by probing with anti-Uba3 and anti-NEDD8 antibodies. Numbers indicate relative density of the bands quantified by scanning densitometry, normalized to β-Actin and are presented relative to unstimulated cells (0 h) or uninfected mice (day 0). Data are representative of three independent experiments with similar results.