Fig. 3

PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a, b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c, d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K^KR in the presence of [γ-³²p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K^KR-GFP. C>N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C < N: higher Myc signal intensity in nucleus than in cytoplasm. n = 100 cells were examined for each experimental condition. i 3XSd2-luc reporter assay in S2 cells transfected with the indicated constructs. Data are means ± s.d. from three independent experiments. j Drosophila adult eyes expressing the indicated Yki transgenes in the absence or presence of UAS-PRP4K. Quantification of eye size for the indicated genotypes. Data are means ± s.d. from three independent experiments. N ≥ 5 for each genotype. k S2 cells transfected with the indicated constructs were treated with or without LMB. Cell extracts were fractionated into nuclear and cytosolic fractions, followed by western blot analysis with the indicated antibodies. H3: histone 3. Tub: tubulin. l S2 cells were transfected with the indicated constructs. Cell lysates were immunoprecipitated with a Myc antibody, followed by western blot analysis with HA and Myc antibodies, or directly subjected to western blot analysis with GFP and HA antibodies. Scale bars, 10 μm (g)