Fig. 4: Colocalization of JAK2 with CHK2 during mitosis.

A–C Costaining of JAK2 and γ-tubulin at the centrosome in mitotic cells. Parental or CHK2 KO HeLa cells were treated with nocodazole overnight, without release (prometaphase) or released for 1 h (metaphase), and costained with anti-γ-tubulin (a centrosome marker), anti-JAK2, and anti-CHK2 antibodies (A). Proportion of cells with JAK2 localized at the centrosome was calculated and their overlapping coefficients with γ-tubulin were measured and shown in (B), (C), respectively. Data were collected from three independent experiments, each of 40–50 cells. D, E Costaining of JAK2 and CHK2 pY156 at centrosomes. Confocal microscopy was done as described above except that the CHK2 pY156 antibody was used (D). Overlapping coefficients between JAK2, γ-tubulin, and CHK2 pY156 staining are analyzed using Zen Black 3.0 SR and shown in (E). F CHK2 pY156 staining was significantly reduced in cells treated with the JAK2 inhibitor. Averaged centrosomal staining intensity from 17 to 20 cells is shown as a percent of untreated control. Mean ± SD from three independent experiments are displayed. Numbers in the scatter plots (C), (E) represent cell counts. Scale bar, 5 μM.