Fig. 2.

FBXL5 regulates IRP2 ubiquitination and iron-dependent degradation. (A) Flp-In TREx-293 cells stably expressing HA-FLAG-FBXL5 or control cells were treated for 8 hours with FAC or DFO. WCEs were probed with antibodies to FLAG, IRP2, ferritin, and β-tubulin. (B) HEK293 cells were transfected with nonspecific or FBXL5 siRNAs and then treated with or without FAC for 8 hours. WCEs were immunoblotted with the specified antibodies. (C) Flp-In TREx-293 cells stably expressing HA-FLAG-FBXL5 or HA-FLAG-FBXL5-ΔF-box or control cells were pulsed with ³⁵S-met/cys for 1 hour and then chased in medium supplemented with or without additional FAC. ³⁵S-labeled endogenous IRP2 was immunoprecipitated with antibody to IRP2 and half-lives (t 1/2) are shown as average ± SD (n = 2 independent experiments). (D) Flp-In TREx-293 cells stably expressing FLAG-IRP2 were treated with nonspecific or FBXL5 siRNAs, pulsed with doxycycline overnight so as to induce FLAG-IRP2 expression, and then chased in medium supplemented with FAC. WCEs were probed with antibodies to FLAG and actin. FLAG-IRP2 half-lives (t 1/2) are shown as average ± SEM (n = 3 independent experiments). (E) HEK293 cells were cotransfected with HA-Ub, FLAG-IRP2 and Myc-FBXL5, Myc-FBXL5-ΔF-box, or empty vector and then treated with FAC and MG132 for 4 hours and ubiquitin conjugates immunoprecipitated by using antibodies to HA. HA-immunoprecipitates (IP: HA) and WCEs were immunoblotted with antibodies to FLAG, c-Myc, and β-tubulin.