Immunofluorescence Labeling Protocol

Immunofluorescence was performed as previously described [17 (link),19 (link)]. Cells were fixed in 4% EM-grade formaldehyde in PEM buffer (80 mM potassium PIPES [pH 6.8], 5 mM EGTA, and 2 mM MgCl₂) and quenched with 0.1 M ammonium chloride for 10 min. For samples in which no antibodies are used, cell membranes were permeabilized using 0.5% Triton X-100 for 10 min and DNA stained using DAPI (1 μg/ml) for 10 min. For samples with antibody labeling, permeabilization was with 0.5% Triton X-100 for 30 min. Cells were incubated at room temperature in blotto (5% milk in Tris-buffered saline/Tween 20) for 30 min, and then specific primary antibodies were added overnight at 4°C prior to 1 hr of secondary antibody (Alexa 647 conjugates; Molecular Probes) and DAPI staining (1 μg/ml for 10 min). The primary antibodies used were: mouse anti-Ser5-phospho RNA polymerase II (Abcam, ab5401) and mouse anti-SRC-3 (BD Transduction Labs No. 611105).