Figure 1. β-TrCP1 binds to FBXW2 and negatively regulates FBXW2 levels.

(a) β-TrCP1 binds to endogenous FBXW2: Cell lysates from H1299 cells were pulled down with anti-FBXW2 or anti-β-TrCP1 Abs, followed by IB with indicated Abs. (b) β-TrCP1 binds to phosphor-FBXW2 peptide: Cell lysates from A549 cells were incubated with bead-conjugated FBXW2 non-phosphorylated or phosphor-peptide containing β-TrCP binding motif. Beads were washed and subjected to IB with indicated Abs. (c–e) Overexpression of β-TrCP1, but not its ΔF mutant, decreases the levels of FBXW2 protein: H1299 and H358 cells were co-transfected with F-FBXW2 (FLAG-tagged FBXW2) and increasing amounts of β-TrCP1, or transfected with increasing amounts of β-TrCP1 or β-TrCP1ΔF alone, followed by IB with indicated Abs. The band density was quantified. (f) β-TrCP1 overexpression has no effect on FBXW2 mRNA: H1299 cells were transfected with β-TrCP1 or vector control, followed by qRT-PCR for mRNA expression. Error bars indicate mean +s.d. of three repeats. (g) β-TrCP silencing increases the endogenous levels of FBXW2 protein, but not mRNA: A549 and H23 cells were transfected with siRNA targeting both β-TrCP1 and β-TrCP2, along with scrambled siRNA, followed by IB (top panels) or qRT-PCR (bottom panel). Error bars indicate mean +s.d. of three repeats. (h,i) FBXW2 half-life is shortened by β-TrCP1, but not by β-TrCP1ΔF: FLAG-FBXW2 was transfected into H1299 cells, along with the vector control or plasmid expressing HA-β-TrCP1 or HA-β-TrCP1ΔF. Cells were switched to fresh medium (10% FBS) containing cycloheximide (CHX) 48 h post transfection for indicated time periods and harvested for IB. The band density was quantified. (j) β-TrCP1 RNAi silencing extends protein half-life of endogenous FBXW2. H23 cells were transfected with either control RNAi, or RNAi targeting both β-TrCP1 and β-TrCP2 for 48 h. Cells were cultured in fresh medium containing CHX and incubated for indicated time periods before being harvested for IB.