Goat anti v5

Standard IF procedures were used. Briefly, cells were permeabilized (0.5 % Triton X-100 in PBS) for 15 min and incubated with blocking solution (DAKO #X0909) for 30 min. The following primary antibodies were used: goat anti-V5 (Bethyl #A190-119A) 1:200; mouse anti-FLAG (Sigma #F1804) 1:200; rabbit anti-BRD4 (Abcam #ab128874) 1:200; rabbit anti-TRIM24 (Proteintech #14208-1-AP) 1:400; rabbit anti-K3K23Ac (Cell Signaling #8848) 1:200, and mouse anti-histone H3 (Active Motif #39763) 1:4000. Alexa Fluor-conjugated secondary antibodies were from Invitrogen and Hoechst 33342 (Invitrogen, #H3570) was used for nuclei counterstaining (10 µg/ml). For each Alexa fluorochrome, images (4–10 fields per well; 20X magnifications) were acquired using the Operetta High-Content Screening System (PerkinElmer). Image analysis was performed in Harmony software (PerkinElmer) by selecting nuclei and cytoplasm in Hoechst channel. Gating for single cells of flat morphology was done based on nuclei area, roundness and intensity of Hoechst in the nucleus and in the cytoplasm. In every gated cell, the mean signal intensity value for each fluorochrome was calculated separately in nucleus and cytoplasm. The specific nuclear signal was estimated subtracting mean cytoplasmic value from nuclear; and average value was calculated using at least 1000 cells in each well.