Figure 1

Characterization of monoclonal anti-TIF1β (20A1) and rabbit anti-phosphorylated TIF1β/Ser473 (S473) antibodies. (A) HeLa cell lysate were probed with anti-TIF1β monoclonal antibody, 20A1 (lane 1). Lysate from 293T cells transfected with FLAG-TIF1β were probed with 20A1 antibody (lane 2) or anti-Flag monoclonal antibody, M2 (lane 3). (B) Whole cell extracts were prepared from 293T cells and incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of CIP (30 U) for 30 min at 37°C. Western blotting was performed with antibodies indicated. (C) M2-beads purified FLAG-TIF1β from 293T cells were treated without (lane 1) or with (lane 2) CIP (30U) for 30 min. Western blot was probed with rabbit anti-TIF1β/Ser473 antibody (S473), anti-Flag antibody (M2), and anti-TIF1β antibody (20A1). Wild-type FLAG-TIF1β (lane 3), FLAG-TIF1β/S473A (lane 4) or FLAG-TIF1β/S473E (lane 5), were immunoprecipitated by M2-beads, resolved in SDS-PAGE, transferred onto membrane and probed with antibodies as indicated.