Fig. 4: Faf interacts with Ft and regulates its protein levels in vitro and in vivo.

a Schematic representation of the Faf constructs used in this study. Numbers denote amino acid position. USP DUB domain is represented in green. b Faf binds to Ft. HA-tagged Ft^ICD was co-expressed with FLAG-tagged GFP or Faf^LD in Drosophila S2 cells. Cells were lysed and lysates were subjected to co-immunoprecipitation using FLAG agarose beads. Lysates were analysed by immunoblot using the indicated antibodies for detection of protein expression and co-purification. Tubulin (Tub) was used as loading control. (n = 3 independent experiments). c, d Faf regulates Ft protein levels in Drosophila S2 cells. c HA-tagged Ft^ICD was expressed in S2 cells in the presence or absence of Faf^LD. 48 h after transfection, cells were lysed and lysates were analysed by immunoblot using the indicated antibodies. Ø represents expression of empty vector. d S2 cells were treated with the indicated dsRNAs 24 h before co-transfection with the indicated constructs. Ft^ICD protein levels were analysed by Western blotting with the indicated antibodies 48 h after cell transfection. GFP and Tubulin (Tub) were used as transfection and loading control, respectively. (n = 3 independent experiments). e–h Faf regulates Ft protein levels in vivo. Shown are XY confocal micrographs of third instar wing imaginal discs expressing the indicated constructs under the control of hh-Gal4, showing Ft antibody staining (e-h; red in e”-h”), Arm antibody staining (e’-h’; cyan in e”-h”), and direct fluorescence from GFP (green in e”-h”). Compared to the controls (e, lacZ^RNAi), faf depletion (f) and faf over-expression (g) resulted in a decrease or increase in Ft protein levels, respectively. Shown is also ft^RNAi control (h) to validate antibody specificity. Ventral is up in XY sections, whilst GFP marks the hh-Gal4-expressing posterior compartment (right). Dashed white line depicts boundary between anterior and posterior compartments. (n = 11, 14, 12, 8). Scale bar: 50 µm.