1. Global analysis of RV[S/T]F motif phosphorylation during mitosis.
Global analysis of RV[S/T]F motif phosphorylation during mitosis. (A) The phosphoepitope-specific antibody generated using the peptide RRVpSFADK recognizes several proteins in immunoblots of HeLa cell lysates (top). Lysates from synchronized HeLa cells at the indicated times after release from T/N block were probed with this RVp[S/T]F (p-RV[S/T]F)–specific antibody to monitor changes in the phosphorylation status of this motif during the cell cycle. Phosphorylation of PP1 at Thr320 (p-Thr320) was also assessed at these time points. Equal loading was confirmed by immunoblotting for tubulin. Mass markers are indicated in kilodaltons. Three replicates of the p-RV[S/T]F immunoblot were quantified using ImageJ software and normalized to the mitotic (0.5 hours) sample (bottom). Error bars represent means ± SD. (B) Immunofluorescence staining of HeLa cells showing 4′,6-diamidino-2-phenylindole (DAPI), tubulin, and p-RV[S/T]F at different phases of the cell cycle. More than 50 cells were imaged for each condition in four independent experiments. Scale bar, 20 μm. a.u., arbitrary units.
Isha Nasa et al., Sci. Signal. 2018;11:eaai8669
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