Figure 3
Thr4 phosphorylated Pol II co-localizes with centrosomes in M phase.
Representative images of a metaphase cells treated with 0.1% Triton X-100 before PFA fixation. (a) HeLa cells were stained with a Thr4-P-specific mAb (6D7, red) and mAb 8WG16 (green) recognizing the CTD of Pol II independently of specific modifications. Co-localisation of Thr4-P and 8WG16 mAbs signals was detected in >98% of mitotic cells. (b) Immunofluorescence image of Rpb3 mAb recognizing the human RNA polymerase II subunit 3 (red) and a centrosome-specific Ab (γ-tubulin; green). Signals from merged images were quantified using Image J 1.37 V and the plug-in RGB profiler. Line scans were used to measure the relative localizations of 8WG16/Rpb3 and Thr4-P-specific signals. (c) Conditional expression of Δ5 mutant in Raji cells. Recombinant polymerase were induced by removal of tetracycline. After 24 h the expression levels of endogenous and recombinant polymerases were analyzed by western blot. The HA mAb allows the visualization of recombinant HA-tagged Pol II (red). Recruitment of Pol II to centrosomes occurs independently of the CTD. Scale bars, 5 μm.
