Figure 4.

Pellino-1 directly interacts with Slug and Snail, and promotes K63-mediated polyubiquitination of Slug and Snail. (a) 293T cells were transfected with TAP (control), TAP-Pellino-1 (Flag-tagging Pellino-1). At 36 h post-transfection, the cells were inspected for GFP expression under microscope (lower) and harvested and isolated through tandem affinity purification (TAP). The bound proteins were immunoblotted with the indicated antibodies (upper). Transfection efficiency was evaluated by inspecting cells expressing GFP under microscope (BF, bight filed; lower). (b) A549 cell lysates were incubated with GST or GST-Pellino-1, and subjected to immunoblotting with the indicated antibodies. (c) GST, GST–Slug or GST–Snail fusion proteins were incubated with purified His-Pellino-1, and subjected to immunoblotting for Slug, Snail and Pellino-1. (d) 293T (upper panel) or A549 (bottom panel) cell lysates were immunoprecipitated with anti-Pellino-1 antibody and immunoblotted with anti-Snail, anti-Slug and anti-Pellino-1 antibodies. Asterisk denotes nonspecific band. (e) HCT116 cells were transfected with Myc-Pellino-1 FL or Myc-Pellino-1-ΔC (ring domain deletion mutant) in combination with HA-Ub and Flag-Slug (left) or Flag-Snail (right). At 36 h post-transfection, the cells were harvested and immunoprecipitated with an anti-Flag antibody. The Slug or Snail protein complexes were subjected to immunoblotting with an anti-HA and anti-Flag antibodies. (f) HCT116 cells were transfected with Myc-Pellino-1, HA-UB, HA-UB K48, HA-Ub K63 and Flag-Slug (left) or Flag-Snail (right) in combination. At 36 h post-transfection, the cells were harvested and immunoprecipitated with an anti-Flag antibody. The Slug or Snail protein complexes were subjected to immunoblotting with anti-HA and anti-Flag antibodies