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. Author manuscript; available in PMC: 2019 Apr 9.
Published in final edited form as: Sci Signal. 2018 May 15;11(530):eaai8669. doi: 10.1126/scisignal.aai8669

Fig. 3. Global analysis of RV[S/T]F motif phosphorylation during mitosis.

Fig. 3.

(A) The phosphoepitope-specific antibody generated using the peptide RRVpSFADK recognizes several proteins in immunoblots of HeLa cell lysates (upper panel). Lysates from synchronized HeLa cells at the indicated times following 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 kDa. Three replicates of the p-RV[S/T]F immunoblot were quantified using ImageJ software and normalized to the mitotic (0.5 h) sample (lower panel). Error bars represent mean ± SD. (B) Immunofluorescence staining of HeLa cells showing DAPI, tubulin, and p-RV[S/T]F at different phases of the cell cycle. >50 cells were imaged for each condition in four independent experiments. Scale bar, 20μm.