Additional file 4.
Figure S3: Immunoblotting and immunofluorescence assays demonstrate decrease in cyclin A protein as cells enter mitosis. (A) HeLa cells were synchronized at G1/S transition with hydroxyurea and later released and samples were collected at 4 h intervals and processed for immunoblotting and immunofluorescence. Cyclin A was visualized with mouse anti-cyclin A antibody in conjugation with anti-mouse Alexa 594 antibody (right panel) while DNA was stained with DAPI (left panel). Representative fields of different time points have been shown. Note that Alexa 594 emission is visible as a red color after passing through the emission filter of Nikon TE2000-S inverted fluorescence microscope. (B) Levels of endogenous cyclin A and Mcm10 were evaluated by specific antibody. ASN refers to samples from asynchronously growing cells while NS points to a non-specific band that displays equal protein loading in different lanes. (C) Flow cytometry of propidium iodide-stained DNA of HeLa cells, as described in (A), shows a G1/S block and release. The colored key denotes cells obtained at different time-points and the FACS histogram shows peaks corresponding to different phases of the cell cycle.
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Kaur et al. BMC Cell Biology 2010 11:84 doi:10.1186/1471-2121-11-84