Anti rb flag f7425

HeLa cells were first transfected with a plasmid carrying a Flag-tagged CENPE fragment (AA2280–2331, or AA2528–2701) for 72 hr, and then treated with 2 mM of thymidine for 19 hr. After synchronization, cells were released from thymidine for 3 hr, and further cultured in the presence of 100 ng/ml of nocodazole for an additional 13 hr. For immunofluorescence staining, cells were prepared as described by Liu et al., (Liu et al., 2003 (link)). Fixed cells were incubated with primary antibodies for at least 60 min. The final concentration for each anitibody was 0.2 µg/ml. Anti-Rb-Flag (F7425) was purchased from Sigma. Anti-γ-tubulin (Ab11316) was obtained from Abcam Inc. Secondary antibodies including Alexa Fluor 488- and Alexa Fluor 555-conjugated goat anti-rabbit, and goat anti-mouse were purchased from Life Technology Inc. and used at 1:200 dilution. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). All images were acquired with an upright epi-fluorescence microscope, Leica DM6000 B (Leica Microsystems Inc) with OpenLab image capturing software (PerkinElmer), analyzed with OpenLab image analysis software.

Anti-Rb IgG negative control (Ab46540), Anti-Ms IgG negative control (Ab81216), Anti-CTCF (Ab70303), anti-CENP-E (Ab5093), Anti-H3K9me3 (Ab8898), anti-H3K9ac (Ab4441), anti-CENP-A (Ab13939), anti-CENP-B (Ab25734), anti-CENP-C ((ab171774) and Anti-γ-tubulin (Ab11316) were purchased from Abcam Inc. Anti-β-Actin (sc-130300) and Anti-Rb Cenp-E (sc 22790) were obtained from Santa Cruz Biotechnology. Anti-Rb-Flag (F7425) was purchased from Sigma.