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Figure 5 | BMC Molecular Biology

Figure 5

From: Phosphorylation at Ser473 regulates heterochromatin protein 1 binding and corepressor function of TIF1beta/KAP1

Figure 5

PKC is involved in the phosphorylation of TIF1β/Ser473 in early S phase. (A) HeLa cells were synchronized with double thymidine block at G1/S phase and treated with various kinase inhibitors before being released for 1 hour to early S-phase (experimental procedures are depicted in the bottom scheme). Lanes 1–6, Western blot of whole cell extracts of cells arrested at G1/S boundary. Lanes 7–12, Western blot of early S-phase extracts. The inhibitors used were CaMK-II inhibitor KN93 (KN, 1 μM), PKC inhibitor Ro-31-8220 (Ro, 100 nM), CK1 inhibitor D4476 (CK-I, 10 μM,), staurosporin (St, 20 nM), and DMSO as a control. The levels of TIF1β (20A1), phosphorylated TIF1β/Ser473 (S473) and α-tubulin are shown. Quantitative results of the Western blots in early S phase (by ImageGauge software) showing the relative TIF1β/Ser473 phosphorylation level in various kinase inhibitor treatment, after normalization to individual TIF1β level. (B) Phosphorylated TIF1β/Ser473 was highly elevated in TPA-treated HeLa cells. Interphase HeLa cells were treated with 100 ng/ml or 250 ng/ml TPA for 1 hour, and whole cell extracts were prepared for Western blot. (C) Phosphorylation of TIF1β/Ser473 in 293T cells was highly induced by over-expressed HA-PKCδ. 293T cells were cotransfected with 1 μg of FLAG-TIF1β plasmid and HA-PKCδ plasmids (1 or 2 μg). FLAG-TIF1β was immunoprecipitated by M2-beads 36 hours post-transfection and probed with antibody to phosphorylated TIF1β/Ser473. Quantitative results (by ImageGauge software) show the relative TIF1β/S473 phosphorylation level in the presence of various amounts of transfected PKCδ plasmid, after normalization to individual TIF1β level. (D) Phosphorylation of TIF1β/Ser473 was specifically increased in HA-PKCδ transfected HeLa cells. HA-PKCδ transfected HeLa cells were fixed 36 hours post-transfection and co-stained with monoclonal anti-HA antibody (red) and TIF1β/S473 antibody (green). DNA was visualized by counter staining with DAPI (blue). Different maginification of images are shown in columns 1–3. The localization of HA-PKCδ or TIF1β in the nucleus is indicated (column 3, arrow). Bars in D represent: column 1, 40 μm; column 2, 20 μm; and column 3, 7 μm.

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