FIGURE 4.

Phosphorylation status of TP domain affects MELK cortex localization. (A) Western Blot analysis of MELK in HeLa cell lysates after 1 h treatment with RO-3306 (Ro) and Roscovitine (Ros) on Nocodazole/MG132 (Noc/MG) arrested cells. Also shown are lysates from asynchronized cells (Asn), thymidine (G1S) or nocodazole (Noc) arrested cells. Cyclin B1 and α-tubulin were also probed to indicate mitotic stage and as a loading control, respectively. (B) GFP fused with wild type (WT) MELK or GFP-MELK-5A (mutant of 5 S/T to A on presumable CDK1 sites) were transfected in HeLa cells expressing mRFP-histone H2A and arrested at prometaphase by treatment with nocodazole and MG132. Single plane still images are shown. Scale bar is 10 µm. (C) HeLa cells were co-transfected with GFP-KA1 domain and mCherry or mCherry-fused different forms of TP domain (mCherry-TP, mCherry-TP-5A, or mCherry-TP-5E), and arrested in prometaphase. Representative single plane still images are shown. Scale bar = 10 µm. (D) Quantification of cortex localized GFP-KA1 signals when different mCherry constructs were co-transfected as in (C). ** denotes P < 0.01 and **** indicated P < 0.0001 (E) Quantification of cortex localized GFP-KA1 signals versus relative GFP/mCherry intensity ratios for all cells quantified in (D).