Figure 3.

FWD1 facilitates IκBα ubiquitination. (A) FWD1 promotes IκBα ubiquitination in vivo. Cells (293T) were transfected with expression plasmids alone (mock; lanes 1 and 4) or plasmids encoding Flag-FWD2 (lanes 2 and 5) or Flag-FWD1 (lanes 3 and 6) in combination with wild-type IκBα (lanes 1 to 3) or 32/36 SA mutant IκBα (lanes 4 to 6). An expression plasmid encoding IKK-2 also was transfected in all lanes. Cell lysates were immunoprecipitated via a Myc tag on wild-type or mutant IκBα, then immunoblotted, and probed with anti-ubiquitin, anti-Myc to detect total IκBα, and anti-Flag to detect FWD1 or FWD2, which is associated with IκBα; 10% of the input lysates was immunoblotted and probed with anti-Flag to show the expression level of FWD1 and FWD2. (B) The F-box of FWD1 is required for ubiquitination but not for association with IκBα. Cells (293T) were transfected with expression plasmids encoding Flag-FWD2 (lane 1), Flag-FWD1 (lane 2), or Flag-FWD1 (ΔF) (lane 3). Expression plasmids encoding Myc-IκBα and IKK-2 were transfected in all lanes. Cell lysates were immunoprecipitated via a Myc tag on IκBα, then immunoblotted, and probed with anti-ubiquitin, anti-Myc to detect whole amount of IκBα, and anti-Flag to detect FWD2, FWD1, or FWD1 (ΔF). (C) In vitro ubiquitination of IκBα is facilitated dramatically by recombinant FWD1. Purified recombinant proteins were generated as described and mixed in combination as indicated with S100 lysate from NIH 3T3 cells. Reaction mixtures were immunoprecipitated with anti-rabbit IgG (lane 1) or anti-IκBα (lanes 2 to 7), then subjected to immunoblotting, and probed with anti-ubiquitin (Upper) or anti-Myc to indicate IκBα and FWD1 in the precipitates (Lower).