Fig. 2.

β-TrCP1 is a substrate of SCF^β-TrCP2 and β-TrCP2 is a substrate of SCF^β-TrCP1. a HEK293 cells were transfected with vector or the indicated FLAG-F-box plasmids for 48 h. Cells were then subjected to IP with FLAG beads and IB with anti-β-TrCP2, CUL1, and FLAG Abs. An asterisk indicates corresponding FLAG-F-box proteins. b Cells were transfected with siRNA targeting β-TrCP1 or β-TrCP2 or with scrambled control siRNA for 48 h and then subjected to IB with anti-β-TrCP1, β-TrCP2, and Actin Abs. c Cells were transfected with the indicated siRNA for 48 h. Then cells were treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, β-TrCP2, and Actin Abs. d Cells with endogenous HA-β-TrCP2 established by CRISPR-Cas9-mediated knock-in were infected with indicated lentiviral shRNA virus for 72 h. Then cells were treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-HA, β-TrCP1, and Actin Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (mean ± S.E.M., n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (c, d, right). e, f HEK293 cells were transfected with the indicated plasmids for 48 h, lysed under denaturing conditions, and then pulled down by Ni-NTA beads. Pull-downs (top) and whole-cell extracts (bottom) were subjected to IB with anti-FLAG, HA, and Actin Abs. WCE: whole-cell extracts