Fig. 2

Downregulation of cIAP1 translation caused the upregulation of NIK protein. A Western blotting analysis of ubiquitinated NIK in HA-tagged ubiquitin (HA-UB)-expressing parental or LM05 cells after treatment with 10 μM MG132 for 4 h. The protein lysates of HA-UB expressed in parental or LM05 cells were immunoprecipitated with an anti-NIK antibody and then immunoblotted with an anti-HA antibody. B Representative western blotting (upper) of cIAP1, 2, TRAF2 and 3 in parental, LM05 and LM1-2–1 cells. qRT-PCR (lower, n = 3, Welch’s t-test) analysis of cIAP1 expression in parental and LM05 cells. C Assessment of cIAP1 protein stability in parental and LM05 cells treated with 10 µg/mL cycloheximide (CHX). Quantification of cIAP1 protein level normalized by α-Tubulin protein level in each time point (n = 3, two-way ANOVA followed by Bonferroni's multiple comparisons test). D. Assessment of nascent cIAP1 protein levels in parental and LM05 cells using a click reaction. Parental and LM05 cells were treated with 50 μM L-homopropargyl glycine (HPG) for 24 h. The nascent proteins labeled with HPG were conjugated to biotin using a click reaction. The biotinylated proteins were purified with streptavidin beads and subjected to western blotting. All data are representative of three independent experiments and are shown as the mean ± SEM. n.s. not significant