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. 2019 Jan 30;15(1):e1007955. doi: 10.1371/journal.pgen.1007955

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© 2019 Misra, Irvine

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 6 — (A-D) Horizontal apical sections of wing imaginal discs expressing en-Gal4 UAS-dcr2 D:GFP UAS-RFP along with UAS-elgi-Myc-HA (UAS-elgi) (A, A’), UAS-elgi^CS-Myc-HA (UAS-elgi^CS) (B, B’), UAS-app-RNAi (C, C’), or UAS-elgi-RNAi and UAS-app-RNAi (D, D’), showing the effect on Dachs:GFP (D:GFP, green) levels and localization in the posterior compartment (marked by RFP, red). Dashed white line marks the A-P compartment boundary. (E-E”) Horizontal apical sections of wing imaginal discs expressing nub-Gal4 UAS-dcr2 UAS-D:RFP UAS-Vam:GFP UAS-elgi-RNAi UAS-app-RNAi showing close up view of D:RFP (red) (E’) and Vam-GFP (green) (E’) colocalization. (F,G) Horizontal apical sections of wing imaginal discs expressing en-Gal4 UAS-elgi^CS D:GFP (F) or along with UAS-app (G) showing the effect on localization of Dachs:GFP (D:GFP) in the posterior compartment (to the right). Dashed white line marks the A-P compartment boundary. Scale bar is 6 μm in A, 33 μm in B-D, 17 μm in E and 19 μm in F and G. (H) Western blot showing results of ubiquitination experiments on proteins co-expressed in S2 cells. 3xFLAG tagged Elgi or Elgi^CS were co-expressed with either BirA or BirA-BioUb and immunoprecipitated using anti-Flag beads. Elgi was detected by anti-FLAG antibody and biotinylated Ubiquitin was detected with IRDYE680 conjugated streptavidin (Red). (I) Western blot showing results of co-immunoprecipitation experiments on proteins co-expressed in S2 cells. V5-tagged Vam or Elgi were co-expressed with full length Elgi-3x-FLAG, and complexes were precipitated using anti FLAG affinity resin and immunoblotted with anti-V5 and anti-FLAG antibodies. GAPDH was used as a control for loading and transfer in the input lanes. Elgi-3x-FLAG has a slower mobility compared to Elgi-V5 due to the larger epitope tag. (J) Western blot showing results of co-immunoprecipitation experiments on proteins co-expressed in S2 cells. V5-tagged Vam, App (App-V5) or Fat-ICD (Fat-ICD-V5) were co-expressed with Elgi-3x-FLAG, and complexes were precipitated using anti-FLAG affinity resin and then immunoblotted with anti-V5 and anti-FLAG antibodies. GAPDH was used as a control for loading and transfer in the input lanes. Arrow points to the full length App in the input lane.