FIGURE 4.

FBXL5 degradation under iron-deplete conditions requires functional ubiquitin-proteasome system and regulatory sequence. A, immunoblot analysis of immunoprecipitated (IP) FLAG-FBXL5 from stably transfected HEK 293T cells treated with FAC or DFO in the absence or presence of the proteasome inhibitor MG132. B, ubiquitination of FBXL5 Hr is not dependent on FBXL5. HEK 293 cells stably expressing the FBXL5 Hr domain were transfected with a negative control siRNA (NT) or siRNA targeting endogenous FBXL5 prior to treatment with FAC or DFO in the absence or presence of MG132. C, immunoblot analysis of FLAG-Hr-HA accumulation in ts-20 BALB/c 3T3 cells incubated with FAC or DFO for 6 h followed by a switch to either E1-permissive (35 °C) or E1-restrictive (39 °C) temperatures for 24 h in medium containing FAC or DFO as indicated. D, immunoblot analysis of FLAG-Hr-HA accumulation in ts-20 BALB/c 3T3 cells. Cells were incubated with FAC for 6 h and maintained in 1 or 21% O₂ at 35 °C or 39 °C for 24 h. E and F, immunoblot analysis of FBXL5 accumulation from expression constructs having increasingly longer N-terminal deletions (E) or short internal deletions (F). FL, full length.