Figure 4
Knockdown of Pol II interactors RUVBL1 and UXT abolishes Thr4-P levels at centrosomes.
(a) The interactome of Pol II. Volcano plot displaying the significant proteins interacting with the recombinant Rpb1 (αHA) when compared to the controls (Pes1 and Bop1). Thresholds: Log 2 Fold change ≥ 5; p-value < 0.01. Indicated are the 12 subunits of Pol II subunits (red) and the 11 subunits of the non-canonical R2TP-Prefoldin-like complex (green). Data based on three biological independent replicates. (b) Western blot analysis of extracts from HeLa cells 48 h after siRNA knockdown of RUVBL1 and UXT. GAPDH3 served as a loading control. (c) Signals for Thr4-P (6D7, red) as well as RUVBL1/UXT (green) co-localized in each phase of mitosis. Representative images of metaphase and anaphase chromosomes are shown. Immunofluorescence images of UXT (green) and Thr4-P-specific (red) mAb in HeLa cells 48 h after siRNA knockdown. >30 cells were analyzed for each experiment; 13% of mitotic cells show loss of the signal for UXT (or RUVBL1) at centrosomes accompanied by the loss of the signal for Thr4-P. Line scans were used to measure the relative localizations of RUVBL1, UXT and Thr4-P-specific signals. Scale bars, 5 μm.
