Figure 3.
JNK specifically phosphorylates YAP in vivo. (a) HaCaT, U2OS, MCF7 and 293T cell lines were treated with anisomycin or DMSO (control) for 60 min before harvesting. YAP band shift was analyzed as described before and lysates were analyzed by Western blotting with the indicated antibodies. (b) Upper panel: U2OS cells were treated with SP600125 or an equivalent volume of DMSO for 3 h, followed by treatment for 15 min with anisomycin or DMSO. Lower panel: HaCaT cells were treated with SB203580 or DMSO for 3 h prior followed by a 15 min treatment with anisomycin or DMSO, harvested and lysates were analyzed using the indicated antibodies. (c) U2OS cells were transfected with Flag–YAP and either siControl (siCTL) or siJNK1 and siJNK2 (siJNK). The cells were then treated with anisomycin for 1 h and subsequently Flag–YAP was immunoprecipitated from lysates. IP inputs and eluates were analyzed by Western blotting using the indicated antibodies. (d) BWT cells were transfected with Flag–YAP, Flag–mut2AYAP and Flag–mut2DYAP. The cells were irradiated with 30 J/m2 UV-C and harvested 1 h later for Western blot analysis with the indicated antibodies. (e) HaCaT cells were irradiated with 50 J/m2 UV-C and harvested at 10, 20, 30 min and 2 h. Endogenous YAP was immunoprecipitated and IP inputs and eluates were analyzed by Western blotting using the indicated antibodies