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. 2020 Sep 1;10(9):2785–2799.

Figure 4.

Figure 4

ASK1 binds to the WW domain of YAP and TAZ. (A) A structural sketch of the YAP and TAZ and their WW domains. Tead-BD, Tead binding domains; WW, WW domain; WW1, the first WW domain; WW2, the second WW domain; TAD, Tead activating domain; PDZ-BD, PDZ binding domain. The numbers labeled in YAP and TAZ constructs indicate the amino acid residue positions. (B) ASK1 binds to the WW domain of YAP and TAZ. The GST fused WW domain was expressed in bacteria, and purified with the glutathione-conjugated agarose beads. The bead-bound GST-WW domain was incubated with the ASK1-expressed HEK293T cell lysates. The co-precipitated ASK1 was detected by immunoblotting with an anti-ASK1 antibody. (C and D) The amino acid residues 799-PPFY-882 in ASK1 is not the YAP- or TAZ-binding site. The GST-WW domain pulldown assay was performed the same as in (B) except the bead-bound GST-WW domain was also incubated with the HEK293T cell lysates containing the ASK1 mutant ASK1-Y882A in which the 799-PPFY-882 is mutated into 799-PPFA-882. (C) The GST-YAP-WW domain pulldown assay; (D) The GST-TAZ-WW domain pulldown assay.