rabbit polyclonal anti rcc2 antibodies  (Novus Biologicals)

Journal: Nature Communications

Article Title: TD-60 links RalA GTPase function to the CPC in mitosis

doi: 10.1038/ncomms8678

Figure Lengend Snippet: ( a ) Immunoblots of whole HeLa cell extract (WCE) transfected with siRNAs targeting two different regions of human TD-60. Immunoblot for RCC2 (TD-60) and RalA, with β-actin as a loading control. ( b ) Mitotic index of TD-60-depleted cells 48 h after transfection with siRNA is increased from prophase to metaphase. Graphs report the mean±s.e.m. ( n =4). ( c ) Control and TD-60-depleted cells fixed with 4% PFA were stained with DAPI (blue), α-tubulin (red) and aurora B (green). Scale bar, 10 μm. ( d ) The proportion of prometaphase cells with abnormal spindle shapes (in which the poles were partly separated but nascent spindles were confined within the regions containing chromosomes) in TD-60-depleted and control cultures was determined by a blinded observer ( n =4). In this and all subsequent figures, graphs report the mean±s.e.m., with statistical significance assessed using the Mann–Whitney U -test (* P <0.05). ( e ) Both prometaphase and metaphase cells show an increased level of centromeric Aurora B after 48 h TD-60 depletion. TD-60-depleted cells were fixed with 4% PFA and stained with DAPI (blue), ACA (red) and Aurora B (green). Scale bar, 10 μm. ( f ) Quantification of Aurora B intensity from the experiment illustrated in e using ImageJ and normalized to the ACA control ( n =15, ** P <0.005 by Mann–Whitney U -test). More information regarding quantification can be found in . DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Immunoblotting analysis was performed using the following primary antibodies: anti-RCC2 rabbit polyclonal antibody (Cell Signalling 1:100–1:200), anti-RalA mouse monoclonal antibody (BD Bioscience 1:100), anti-Ran mouse monoclonal antibody (BD Bioscience 1:1,000), anti-Tubulin B512 mouse monoclonal antibody (Sigma 1:10,000), anti-Beta Actin mouse monoclonal antibody (Sigma 1:1,000) and anti-GFP rabbit polyclonal (Life Technology, 1 μg ml −1 ).

Techniques: Western Blot, Transfection, Control, Staining, MANN-WHITNEY