Figure 6.

Itch attenuates WBP2 protein stability and associates with WBP2 in CD4 T cells. (A) Naïve CD4 T cells fromIL4 KO or Itch IL4 DKOmice were harvested and cultured in complete media for 6 h with no stimulation. Cells were either left untreated, or treated with the indicated drugs (CHX = cycloheximide). Western blots were performed on whole cell lysates, and membranes were probed with anti-WBP2 (rabbit polyclonal) and anti-β-actin (mouse polyclonal), and anti-rabbit 800 and anti-mouse 680 secondary antibodies were used. (B) Quantification of three biological replicates across three experiments described in (A). One additional experiment was performed on untreated cells. Each sample was normalized to β-actin, and these values were normalized to the untreated IL4 KO control sample within each experiment. p = 0.0003 (untreated), p = 0.004 (CHX-treated), p = 0.070 (MG132 and chloroquine-treated) based on unpaired t-test after the described normalizations. (C) Naïve CD4 T cells were isolated as described and activated with plate-bound anti-CD3 and anti-CD28 antibodies for 24 h in IL-2-containing media. After 18 h of activation, cells were either harvested and pelleted, left untreated, or treated with the indicated drugs for 6 h (between 18 h and 24 h). Western blots were performed as described in (A). (D) Quantification of five biological replicates in five experiments described in (C). Each sample is normalized to β-actin. Each value was normalized to its own 18 h timepoint. p-values were determined by unpaired t-test. p = 0.0009 (CHX-treated). All error bars represent mean ± SEM. (E-F) IL4 KO and Itch IL4 DKO cells were transfected with either control siRNA or WBP2 siRNA and collected at the indicated timepoints to check for protein knockdown (E) and (F) or RNA knockdown (F). n = 3 biological replicates across three independent experiments for each protein and RNA. In one of the three biological replicates, RNA and protein were analyzed in the same experiment. Data were analyzed by paired t-test, pairing IL4 KO cells with Itch IL4 DKO cells from the same knockdown experiment. WBP2 protein or RNA was normalized to β-actin protein or RNA. These values were then normalized to the relative WBP2 abundance in control-siRNA-treated cells at the same timepoint. p = 0.5641 (RNA), p = 0.0043 (protein). (G) Representative immunoprecipitation of WBP2 in CD4 T cells. After 20 h of activation, IL4 KO or Itch IL4 DKO cells were treated with MG132 and chloroquine. Cell pellets were lysed, precleared, and incubated overnight with either WBP2 antibody or Rabbit IgG control, as indicated. Pulldown was run on a gel and blotted with anti-Itch and anti-WBP2 antibody and corresponding secondary antibodies as described in (A). Blot is representative of two biological replicates done in independent experiments.