TAK1 phosphorylation and activation by TAB4. A,
combinations of FLAG-TAK1, T7-TAB1, HA-TAB4, and HA-α-4 were expressed
in HEK293T cells and analyzed by immunoblotting with (top to
bottom) 1) anti-(Thr(P)184/Thr(P)187) TAK1, 2)
anti-FLAG, 3) anti-(Ser(P)177/Ser(P)181) IKKβ, and
4) anti-HA (bottom panel). Lanes 1–3 show extracts
from cells co-expressing 1) FLAG-TAK1, 2) T7-TAB1, or 3) FLAG-TAK1 and
T7-TAB1. Lanes 4–6 show extracts from cells co-expressing
HA-TAB4 with FLAG-TAK1 and/or T7-TAB1. Lanes 7–9 show extracts
from cells co-expressing HA-α-4 with FLAG-TAK1 and/or T7-TAB1.
B, phosphatase treatment of FLAG-TAK1 and T7-TAB1. Proteins were
co-expressed without (lanes 1–3) and with HA-TAB4 (lanes
4–6) in HEK293T cells, immunoprecipitated using anti-FLAG M2 beads,
and immunoblotted with anti-T7 (upper panel) and anti-FLAG (lower
panel). Immunoprecipitates (IPs) were treated with vehicle,
1×, or 5× amounts of recombinant MBP-λ-phosphatase
(PPase) as indicated. P-, phospho-.