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1.
Figure 2

Figure 2. From: Wbp2 cooperates with Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo pathway.

Yorkie and Wbp2 physically interact in a WW domain- and PY motif-dependent manner. (a) HA-tagged wild-type and mutant Yki proteins were assessed for their ability to bind to Myc-tagged Wbp2. Immunoprecipitations were performed with rabbit anti-HA antibody and immunoblotting was carried out with mouse anti-Myc to detect Myc-tagged Wbp2 and rat anti-HA to detect HA-Yki. (b) Myc-tagged wild-type and PY motif-mutant Wbp2 proteins were assessed for their ability to bind wild-type HA-Yki. Molecular mass markers (in kDa) and immunoglobulin (Ig) bands are indicated

X Zhang, et al. Cell Death Differ. 2011 August;18(8):1346-1355.
2.
Figure 3

Figure 3. From: Wbp2 cooperates with Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo pathway.

Wbp2 promotes Yorkie's transcriptional co-activator properties. (a and b) Luciferase assays measuring Sd-dependent transcriptional activity in S2 cells expressing the indicated plasmids. The effect of wild-type Wbp2 on wild-type Yki, WW domain mutant-Yki (Yki-WW1*2*) or Yki-S168A was assessed in a. The ability of wild-type Wbp2 or Wbp2 carrying mutations in PY motif 1, PY motif 2 or both PY motifs, to enhance Yki's ability to activate Sd was assessed in b. (c) Luciferase assay in S2 cells expressing the indicated plasmids, measuring Wbp2's ability to activate Yki-Gal4. Error bars in ac represent S.D., n=3

X Zhang, et al. Cell Death Differ. 2011 August;18(8):1346-1355.
3.
Figure 6

Figure 6. From: Wbp2 cooperates with Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo pathway.

Wbp2 subcellular localisation is not altered by the SWH pathway and Wbp2 does not interfere with SWH pathway-mediated phosphorylation of Yorkie. (a and b) Expression and localisation of Wbp2 in tissue lacking wtsX1 were assessed in third instar larval discs. Wbp2 protein expression (red in a and b) is shown in mosaic eye discs with GFP-negative hsFLP wtsX1 clones (aa″) and wing discs with GFP-positive hsFLP/MARCM wtsX1 clones (bb″). Protein and GFP expression images are merged in a″ and b″. Scale bars represent 50 μM. (c) The effect of Wbp2 on SWH-induced Yki phosphorylation was assessed by immunoblotting protein extracts derived from S2 cells transfected with control vector (CON); Wbp2, Yki, Yki and Wbp2; Yki, Sav, Wts and Hpo; or Yki, Wbp2, Sav, Wts and Hpo. Lysates were probed with antibodies against phospho-Yki-S168, total Yki and Tubulin

X Zhang, et al. Cell Death Differ. 2011 August;18(8):1346-1355.
4.
Figure 1

Figure 1. From: Wbp2 cooperates with Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo pathway.

Wbp2 is broadly expressed in larval tissues and is both nuclear and cytoplasmic. (A) Schematic representation of proteins encoded by the wbp2 gene. Two protein products are predicted (337 and 427 amino-acids long) that each contain a GRAM domain and two evolutionarily conserved PY motifs. (B) Wbp2 protein expression assessed by immunoblotting in S2 cells and third instar larval tissues. S2 cells were transfected with vector control (Con), short Wbp2 isoform (Wbp2 – 337) or treated with dsRNA for Wbp2 in the presence or absence of Cellfectin. Tissues assessed were wings, eyes, brains and salivary glands. Wings expressing the wbp2-RNAi 1 transgene under the control of the 32B-Gal4 driver (Wing RNAi) were also assessed. (C) Subcellular localisation of Wbp2 (green) in S2 cells transfected with a Myc-tagged version of the short Wbp2 isoform. Nuclei are marked by DAPI (magenta). (D) The level of Wbp2 knockdown caused by expression of wbp2-RNAi 1 was assessed in vivo. Planar (a and b) and cross (c and d) sections of Wbp2 protein expression (red) in third instar larval wing discs expressing wbp2-RNAi 1 under the control of the en-GAL4 driver. Reduced Wbp2 expression was observed specifically in the posterior compartment of the wing disc, labelled with GFP (green)

X Zhang, et al. Cell Death Differ. 2011 August;18(8):1346-1355.
5.
Figure 4

Figure 4. From: Wbp2 cooperates with Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo pathway.

Wbp2 depletion restricts wing growth and partially rescues lethality caused by deficiency of the warts gene. (a and b) RNAi-mediated depletion of wbp2 in the posterior compartment of the wing (using hh-GAL4) results in reduced wing size (b) compared with wbp2-RNAi control wings that lack the hh-GAL4 driver (a). Flies were reared at 29°C to increase GAL4/UAS expression. (c) Quantification of the effect of Wbp2 knockdown in the hh-GAL4 expression domain in adult wings. wbp2-RNAi 1; hh-GAL4 wings showed a 23% reduction in the posterior compartment of the adult wing (right column) compared with wbp2-RNAi 1 flies that lacked the hh-GAL4 driver (left column). The posterior compartment of the indicated genotypes was measured (in pixels) and compared using an unpaired Student's t-test. Values represent the Mean±S.E.M., ***P<0.0001. (d) The effect of Wbp2 knockdown on lethality caused by the generation of wtsX1 mutant eye discs was assessed. Lethal pupae harbouring wtsX1 mutant eye discs generated using eyFLP/MARCM are shown on the left, and a live adult generated using eyFLP/MARCM harbouring wtsX1 mutant eyes that also expressed wbp2-RNAi 1 is shown on the right. (e) Rescue of animals with wtsX1 mutant eye tissues from late larval lethality to the pharate adult stage of development by two independent wbp2-RNAi lines. Percentage of pupae of the indicated genotypes that developed to pharate adults is shown, with error bars representing S.E.M.. In c and e the number of animals (n) assessed for each genotype is indicated

X Zhang, et al. Cell Death Differ. 2011 August;18(8):1346-1355.
6.
Figure 5

Figure 5. From: Wbp2 cooperates with Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo pathway.

Wbp2 depletion partially reverts overgrowth, differentiation defects and ectopic CycE expression of eye tissue lacking the warts gene. (ac″) The effect of Wbp2 knockdown on the overgrowth of wtsX1 mutant tissue was analysed in third instar larval eye discs with GFP-positive eyFLP/MARCM clones of FRT82B (aa″), wtsX1 (bb″) and wbp2-RNAi 1; wtsX1 (cc″). Discs were stained for F-Actin (red in a, b and c). Control and mutant clones express GFP (green in a′, b′ and c′) and are merged with F-Actin staining images in a″, b″ and c″. Inset images in a, b and c are high magnification views of F-Actin. Arrowheads in a and c indicate the morphogenetic furrow, which was undetectable in b. Knockdown of Wbp2 in wtsX1 clones resulted in reduced overgrowth, a partial return to wild-type F-Actin staining and an increased amount of GFP-negative wild-type tissue. (de″) CycE expression (red in d and e) and the merged images (d″ and e″) were assessed in third instar larval eye discs with genotypes wtsX1 (dd″) or wbp2-RNAi 1; wtsX1 (ee″). In de″ arrowheads indicate the morphogenetic furrow, whereas arrows in dd″ indicate a large wtsX1 clone with elevated CycE. Scale bars represent 50 μM in ae″. (fh) The specificity of Wbp2's role as a regulator of tissue growth was determined by assessing the effect of Wbp2 knockdown on tissue lacking the Tsc1 gene. Eyes from the following adult female flies are shown: (f) eyFLP/MARCM control containing wild-type clones; (g) eyFLP/MARCM generated clones of Tsc1Q87X; (h) eyFLP/MARCM generated clones of UAS-wbp2 RNAi 1; Tsc1Q87X. Expression of UAS-wbp2 RNAi did not suppress eye overgrowth caused by Tsc1 deficiency. Anterior is to the right in all images

X Zhang, et al. Cell Death Differ. 2011 August;18(8):1346-1355.

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