A. In vitro kinase assay and dephosphorylation assay was performed as described in methods. The recombinant GST-YAP2 proteins were incubated with immunoprecipitated LATS2 in the presence of cold ATP at 30°C for 30 minutes. The reaction products were analyzed by immunoblotting with anti-p-S127-YAP2 antibody. The kinase reaction mixture was incubated with the recombinant GST-PP1A at 37°C for 1 hour and followed by Western blotting with anti-p-S127-YAP2 antibody. PP1 dephosphorylates YAP2 at S127 in vitro. B. Lysates of HeLa cells transfected with Myc-YAP2 together with FLAG-PP1A or the control vector were immunoblotted with the indicated antibodies. C. Lysates of cells transfected with FLAG-PP1A or the control vector were blotted with anti-p-S127-YAP2 antibody. PP1A dephosphorylates endogenous YAP2 at serine 127. D. HeLa cells were transfected with plasmid encoding Myc-YAP2, and then treated with PP1A inhibitor OA for indicated time periods. The serine 127 phosphorylation levels of YAP2 were determined by Western blotting. E. HeLa cells were treated with OA for four hours and the cell lysates were analyzed with anti-YAP2 antibody.

A. HeLa cells were transfected with the expression plasmid encoding YAP2 or PP1A plus the 3*Sd-luc luciferase reporter and renilla plasmids. The cell lysates were subjected to dual luciferase assay (t-test; n = 3, p<0.01). OA indicates that HeLa cells were treated with 100 nM OA for 4 hours. PP1A acts synergistically to activate YAP2-mediated transactivation (t-test; n = 3, p<0.01). B. Total RNA was isolated from HeLa cells transfected with the plasmids as indicated. The expression of CTGF was determined by relative quantitative RT-PCR and normalized to GAPDH. PP1A increases the expression of CTGF (t-test; n = 3, p<0.01). C. A2780 cells were transfected with YAP2 or YAP2-S127A plasmid together with or without PP1A. Cell death was analyzed after 24-hour cisplatin treatment followed by flowcytometry (t-test; n = 3, p<0.05). PP1A acts synergistically with YAP2 to enhance ovarian cancer cell survival. D. Lysates of cells infected with adenovirus YAP2 or the control vector treated cisplatin together with or without OA were immunoblotted with the antibody for cleaved caspase 3, p-S127-YAP2, YAP2 or Erk1/2, respectively. E. A2780 cells were infected with the adenovirus encoding YAP2 shRNA or control vector. Cell death was analyzed by flowcytometry after cisplatin treatment for 24 hours together with or without OA (100 nM) for 4 hours before harvesting (t-test; n = 3, p>0.05). F. Cell lysates from (E) were immunoblotted with anti-cleaved caspase 3 or YAP2 antibody.

A. Tandem affinity purification of YAP2 complex was performed as described in methods. The final HA-eluates from cytoplasmic and nuclear fractions were resolved on SDS-PAGE gels and silver-stained. B. PP1A was identified from the mass spectrometry analyses of both cytoplasmic and nuclear HA-eluates. The number of peptide hits for PP1A and several known YAP-interacting proteins are shown. C. Cytoplasmic and nuclear eluates were separated by 10%–40% glycerol gradient gels followed by Western blotting with anti-PP1 antibody. D. GST-pull down was carried out by incubating the recombinant GST-YAP2 or GST alone proteins with lysates of cells transfected with FLAG-PP1A and followed by immunoblotting with anti-FLAG antibody. E. Myc-immunoprecipitates from HeLa cells transfected Myc-YAP2 together with FLAG-PP1A were immunoblotted with anti-FLAG antibody. 2% input was blotted with anti- FLAG, Myc or actin. F. Myc-YAP2 plasmid or the control vector was transfected in HeLa cells and cell lysates were immunoprecipitated with anti-Myc antibody followed by immunoblotting with anti-PP1 antibody. 2% input was blotted with anti- PP1, Myc or GAPDH antibody, respectively. G. Endogenous interaction between YAP2 and PP1A.

A. Myc-immunoprecipitates of HeLa cells transfected with Myc-YAP2 together with PP1A or control vector were immunoblotted with anti-14-3-3β antibody. PP1A expression inhibits the interaction between YAP2 and 14-3-3. B. Myc-immunoprecipitates of HeLa cells transfected with Myc-YAP2 plasmid or control vector and treated with or without OA were blotted with anti-14-3-3β antibody. Phosphatase inhibition increases the interaction between YAP2 and 14-3-3 proteins. C. Nuclear and cytoplasmic fractionation was performed from HeLa cells transfected with PP1A or control vector. The fractionated lysates were immunoblotted with anti-YAP2, GAPDH or Histone H3 antibody. PP1A increases the nuclear levels of YAP2. D. HeLa cells were transfected with the GFP-YAP2 and treated with or without 100 nM OA for 4 hours followed by fluorescent microscopy analysis. E. PP1A increases the nuclear accumulation of YAP2 and OA treatment induced YAP2 cytoplasmic translocation (t-test; n = 3, p<0.01). Data are represented as mean ± SEM. F. HeLa cells were transfected with Myc-tagged YAP2 together with FLAG-tagged PP1A expression plasmid or the control vector. 24 hours after transfection, cells were treated with 50 µg/ml Cycloheximide (CHX) for different time periods. Equal amounts of total protein lysates were subjected to immunoblotting. G. PP1A expression stabilized the protein level of YAP2.