CNOT4 affects PAF1 independent of chromatin binding.

<p>(A) HEK293 cells were fractionated into chromatin-unbound (U) and bound (B) fractions, and the chromatin association of the proteins was determined via Western blotting. GAPHD and H3 were used as markers of the chromatin-unbound and bound fractions, respectively. (B, C) Chromatin-unbound and bound PAF1 was determined by immunoblotting in CNOT4-overexpressed (B) and silenced (C) cells. (D) WT or Myc-mPAF1 Δ285-355-transfected HEK293 cells were treated with MG132 (10 μM) for 6 hr, and Myc-tagged PAF1 protein levels in whole-cell lysates were detected. (E) WT or Myc-mPAF1 Δ285–355 was transfected along with control or CNOT4 siRNA. Myc-tagged PAF1 protein levels in whole-cell lysates were determined using an antibody against Myc. The pGFP plasmid was included as a transfection control. CNOT4 depletion was measured by RT-PCR.