IKKβ and IKKε phosphorylate YAP. ( a ) 293T cells were co-transfected with <t>GFP-YAP</t> and Flag-IKKα, HA-IKKβ or IKKε. The cell lysates were analyzed by western blot with indicated antibodies. The asterisk showed the shifted band ( b , c ) 293T cells were co-transfected with GFP-YAP and HA-IKKβ or IKKε. Cell lysates were immunoprecipitated with indicated antibodies and analyzed by western blot. ( d ) 293T cells were transfected with GFP-YAP and HA-IKKβ or IKKε. The cell lysates were analyzed by phos-tag gel western blot. The asterisk showed the phosphorylated band. ( e ) The recombinant GST-YAP protein was incubated with immunoprecipitated HA-IKKβ or IKKε in phosphorylation buffer. Reactions were subjected to electrophoresis and immunoblotted with antibody against pan-phosphorylated serine/threonine (S/T). ( f ) MCF7 cells were treated with TNFα for indicated times, the cells were then lysed for phos-tag gel western blot. The asterisk showed the phosphorylated band. ( g ) 293T cells were transfected with GFP-YAP and HA-IKKβ/ε. Cell lysates were immunoprecipitated with GFP antibody followed by immunoblotting with <t>TEAD4</t> antibody. ( h ) MCF7 cells were treated with 10 μ M TPCA-1 or 10 μ M IKK-16 for 12 h. Then endogenous YAP was immunoprecipitated and followed by immunoblotting with TEAD4 antibody.
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