Figure 2

Phosphorylation of TIF1β/Ser473 is dynamically regulated during cell cycle progression. (A) HeLa cells were arrested at G1/S boundary by double thymidine block and released into S phase for three hours. The levels of TIF1β and phosphorylated TIF1β/Ser473 were probed with 20A1 and S473 antibodies. The protein levels of cyclin A, cyclin B1, or α-tubulin were also shown. (B) HeLa cells were synchronized by nocodazole (1 μM) treatment for 16 hours. Mitotic shake off cells were collected and released for 8 hours. The levels of TIF1β and phosphorylated TIF1β/Ser473 were probed with 20A1 and S473 antibodies. The protein levels of cyclin B1, phosphorylated Histone H3 S10 (H3S10), or α-tubulin were also probed as indicated. The relative phosphorylation level of TIF1β/Ser473 was compared by quantitative determination of the images from Western blots (by ImageGauge software, normalized to individual TIF1β level) at each time point (shown in the lower panel). (C) HeLa cells were arrested at G1 phase by serum starvation for three days and released to S phase by serum addition. The levels of TIF1β and phosphorylated TIF1β/Ser473 were probed with 20A1 and S473 antibodies. The protein levels of cyclin A, cyclin B1, and α-tubulin were also probed as indicated. HeLa cells at 0 hr (G1) or 2 hours after serum addition (early-S) were immunostained with 20A1 (red) and S473 (green) antibodies, DNA was counter stained with DAPI (blue). (D) Immunostaining of ectopically expressed FLAG-TIF1βs. FLAG-TIF1β/S473A (column 1) or FLAG-TIF1β/S473E (column 2) and wild-type FLAG-TIF1β (column 3) were transiently transfected into HeLa cells. Cells were fixed at 36 hours after transfection and co-stained with M2 (red) and HP1β (green) antibodies. DNA was counter stained with DAPI (blue). Control immunostaining was negative when performed with secondary antibody alone (data not shown). Bars in C represent 30 μm and bars in D show 8 μm.