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. 2016 Jul 20;2(7):e00131. doi: 10.1016/j.heliyon.2016.e00131

Fig. 5.

Fig. 5

Validation of S894 phosphorylation on endogenous INCENP in vivo. (A) HEK293T cells were co-transfected with EGFP-tagged LATS2 and either 6Myc-tagged INCENP-WT, -S894A, or vector alone (vec), and then treated with nocodazole (Noc) and taxol (Tax). Western blotting of these cell lysates was performed with anti-pS894, anti-Myc, and anti-EGFP antibodies. (B) HeLa-S3 cell lysates were treated with λPPase (200 U) with or without protein phosphatase inhibitors. (-) indicates lysates incubated with neither λPPase nor protein phosphatase inhibitors, followed by western blotting with the indicated antibodies. Cyclin B1 is a mitotic marker. α-tubulin is a loading control. Arrowheads indicate bands corresponding to pS894-INCENP. (C) Proteins extracted from HeLa-S3 cells in TNE250 lysis buffer with or without protein phosphatase inhibitors were treated with or without λPPase (200 U), followed by western blotting with the indicated antibodies. Arrowheads indicate the bands corresponding to pS894-INCENP. See Fig. S3 for uncropped western blot images in Figs. 5A–C. Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.