Fig. 6.

WBP2 promotes ubiquitin‐mediated IκBα proteasomal degradation through enhancing BTRC expression. (A, B) Analysis of inhibitor of nuclear factor of kappa beta alpha (IκBα) transcript levels in (A) MDA‐MB‐231 and (B) BT549 cells. WBP2 transcript level was also assessed to confirm efficiency of WBP2 knockdown or overexpression. Data are represented as mean ± SEM, n = 3. **P < 0.01. (C) Cycloheximide chase assay to assess WBP2 effect on IκBα stability. (i) Western blot analysis of IκBα in MDA‐MB‐231 transfected with scrambled (scr) siRNA or pooled WBP2 siRNAs (equal amounts of siWBP2#1&2) and (iii) BT549 transfected with WBP2‐expressing plasmid or vector control. The cells were treated with cycloheximide (CHX) for a duration of up to 90min. β‐Tubulin served as a loading control. (ii, iv) Quantification of IκBα bands in C normalized to β tubulin in (ii) MDA‐MB‐231 and (iv) BT549 cells using imagej software. (D) MDA‐MB‐231 was transfected with scr/pooled WBP2 siRNAs and vector/WBP2 plasmids, as indicated. The cells were serum‐starved overnight and then treated with the indicated proteasomal (red)/lysosomal (blue) inhibitors or DMSO control for 4–6 h before being stimulated with 10 ng·mL⁻¹ of tumor necrosis factor alpha (TNF‐α) for 15 min. (E) In vivo ubiquitination assay performed in MDA‐MB‐231. Cells were treated with MG132 for 4–6 h before His pulldown was performed to immunoprecipitate the ubiquitinated proteins. The total amount of ubiquitinated IκBα was probed in Western blot. (F) WBP2‐silenced MDA‐MB‐231 cells were treated with MG132 (20 μm) for 4–6 h. (i) Western blot analysis was performed to probe the total and phosphorylated IκBα proteins (ii) Relative IκBα phosphorylation was quantified by normalizing phosphorylated IκBα to total IκBα via densitometry analysis. All data in this figure are represented as mean ± SEM, n = 3. **P < 0.01; N.S, nonsignificant. (unpaired t‐test for two group comparisons; one‐way ANOVA followed by post hoc Bonferroni test for multiple group t‐test).