Protein Degradation of RNA Polymerase II-Association Factor 1(PAF1) Is Controlled by CNOT4 and 26S Proteasome
Fig 5
The 255–275 amino acid on PAF1 is required for its degradation.
(A) Schematic diagram of WT and mutant PAF1. “K” designates lysine residues. (B, C) Cells were transfected with Myc-tagged WT or mutant PAF1, and the sub-cellular localization of these proteins was analyzed by immunofluorescence staining with an anti-Myc antibody (green) and DAPI (blue) (B). The percentages of cells containing WT or mutant PAF1 in the “nucleus only” or in the “nucleus and cytoplasm, cytoplasm only” are shown (C). (D) HEK293 cells were transfected with the indicated plasmids for 48 hours. Cells were treated with MG132 (10 μM) for 6 hr, followed by immunoblot analysis. (E) WT PAF1- or mPAF1△255-275-transfected cells were treated with MG132 (10 μM) for 6 hr, and the levels of each PAF1 protein were detected by immunoblot against anti-PAF1. (F) Cells were transfected with WT or mutant PAF1 along with control or CNOT4 siRNA. Top, WCLs were prepared from half of the cells and subjected to Western blot analysis. Bottom, Total RNA was prepared from the remaining cells and analyzed by RT-PCR.