(A) Overexpression of Fbxo6 increases the t1/2 of IκBα only in DKO cells (see Fig S6A–B for WB images). Fbxo6 was overexpressed by transduction with a recombinant lentivirus expressing HAFbxo6. The assay was performed four days after the viral transduction. Average fold increase of IκBα t1/2 by Fbxo6 overexpression in WT and DKO cells are graphed. Error bar: ± SD, n = 3, P value < 0.01.
(B) Overexpression of Fbxo6 in DKO cells reduces degradation of SCF substrates. All samples were analyzed on the same gel and blot, but one lane between WT and DKO samples on the blot image was eliminated and indicated as a space.
(C) Overexpressed Fbxo6 sequesters Cul1 in DKO cells. Cells were infected with recombinant lentiviruses carrying the HAFbox6 gene five days before HAFbxo6 was IP’d from WT3xFLAG-Cul1 and DKO3xFLAG-Cul1 cells in the presence of recombinant Cul1•GSTRbx1 (+ sponge). Equal percent volumes of Input (In), immunoprecipitation eluent (IP), and flow-through (FT) were analyzed by WB. Long (L) and short (S) exposures of endogenous 3xFLAGCul1 are shown. Quantifications of percent Cul1 in the HAFbxo6 IPs are graphed. Error bars: ± SD, n = 3, P value < 0.01.
(D) Fbxo6 overexpression reduces proliferation of DKO cells in a specific, FBP-dependent manner. Cells were treated with recombinant lentiviruses carrying different FBP constructs as indicated. Three days after lentiviral infection, cells were equally seeded and counted every 24 hrs for 4 days. Average cell doubling time is graphed. Error bars: ± SD, n = 3, P value < 0.01. Note that Fbxl16 bound at least as much Skp1 as Fbxo6 but did not bind Cul1 (compare Fig S6G with panel D), and that re-introduction of Cand1 rescued the DKO cells.
(E) Overexpression of Cul1 partially rescues toxicity of overproduced Fbxo6 in DKO cells. Cul1 overexpression was induced by tetracycline. Cell doubling was measured as in (D). Error bars: ± SD, n = 3.
(F) Overexpression of HASkp2 or HASkp2ΔLRR slows cellular proliferation in DKO cells. Cells were infected by lentiviruses and cell doubling was measured as in (D). Error bars: ± SD, n = 3, P value < 0.01.
(G) Overexpression of HASkp2ΔLRR increased the level of apoptosis marker in DKO cells. A representative result of two replicates is shown.