Fig. 3: DCAF1 regulates the degradation of Rheb.

A, B Overexpression of DCAF1 reduces the protein level of Rheb. 293T cells were transfected with different amounts of Flag-DCAF1 and same amount of HA-Rheb (A) or Huh7, HCT116, and EC109 cells were transfected with vector or Flag-DCAF1 (B). C, D Cells were collected and analyzed by IB as indicated. Knockdown of DCAF1 enhances the protein level of Rheb. 293T cells (C) and Huh7, HCT116, and EC109 cells (D) were transfected with siDCAF1 with (C) or without (D) Flag-Rheb, and analyzed by IB as indicated. E Treatment with MLN4924 restores the expression of Rheb in cell lines stably overexpressing DCAF1. Cells with stable overexpression of DCAF1 (Fv-Flag-DCAF1) or its counterpart (Lv-EGFP) were treated with MLN4924 or DMSO, and the protein levels of DCAF1 and Rheb were detected by IB using Flag Ab or Rheb Ab. F DCAF1 promotes Rheb ubiquitination. Cells with stable overexpression of DCAF1 or its counterpart were treated with 10 µM MG132. Rheb polyubiquitination was detected by IP using Rheb Ab and IB with Ub Ab. G Overexpression of DCAF1 decreases Rheb protein stability. Huh7, HCT116, and EC109 cells with stable overexpression of DCAF1 or its counterparts were treated with 100 µg/ml CHX. Cells were harvested at the indicated time points for IB analysis. H Silencing DCAF1 using CRISPR-Cas9 system (sgDCAF1) increases the protein level of Rheb. Cell proteins from sgDCAF1 and sgControl were collected and analyzed using IB. I DCAF1 depletion attenuates Rheb polyubiquitination. sgDCAF1 and sgControl cells were treated with 10 µM MG132, harvested, and subjected to IP with anti-Rheb, followed by IB with anti-Ub. J Silencing DCAF1 increases Rheb protein stability. Huh7, HCT116, and EC109 cells with stable silencing of DCAF1(sgDCAF1) and its counterpart (sgControl) were treated with 100 µg/ml CHX and harvested at the indicated time points for IB analysis. All data were representative of at least three independent experiments (n = 3).