Figure 8.
Ser-138 phosphorylation of IRP1 in response to CIA inhibition. A, wild-type HEK 293 cells were transfected and harvested as in Fig. 1. Endogenous IRP1 was immunoprecipitated from cell lysates using a pan-IRP1 antibody and immunoblotted for Ser-138–phosphorylated IRP1 using Ser-138–phosphospecific IRP1 antibodies (6) (n = 3). B, wild-type HEK 293 cells were transfected with either NT or FBXL5 siRNA for 48 h. During the last 24 h of siRNA treatment, cells were treated with tet to induce the expression of Myc-tagged IRP13C>3S or IRP13C>3S/S138A. Cell viability was determined by trypan blue exclusion. Cell lysates were subjected to Western blotting using antibodies against FBXL5, tubulin, and the Myc epitope on IRP13C>3S or IRP13C>3S/S138A (n = 3). Error bars, S.E.