Figure 2. The CSN positively regulates Ci¹⁵⁵ protein stability in wing discs.

(a) CSN5^null clones are revealed by the absence of GFP (green) in wing discs. Ci¹⁵⁵ stained with the 2A1 antibody (red) is reduced in CSN5^null clones located in the A compartment, with arrow indicating mutant cells in the low-to-intermediate Hh signalling region and arrowhead indicating cells in the low Hh signalling region. (b) GFP-negative CSN4^null clones were generated in wing discs (green). Ci¹⁵⁵ staining (red) is lower in the CSN4^null cells in the A compartment (arrow) than in the wild-type cells. The Ci¹⁵⁵ levels in CSN4^null and wild-type cells located in A/P boundary are similar (arrowhead). (c) CSN4^null clones revealed by the absence of GFP (green) were generated in wing discs that simultaneously express UAS-ci-myc under the control of ms1096-GAL4. Protein levels of Ci-Myc detected by the anti-Myc antibody (red) are reduced in CSN4^null mutant clones (arrow). (d) The ci-lacZ expression (red) is not reduced in CSN4^null mutant clones (arrow) generated in wing discs. (e) GFP-negative CSN5^null clones (green) were generated in wing discs expressing wild-type CSN5 construct under the control of ms1096-GAL4. Expression of wild-type CSN5 rescues the Ci¹⁵⁵ level (red) in CSN5^null clones (arrow). (f) Expression of CSN5^D148N, which loses deneddylation activity, fails to rescues the Ci¹⁵⁵ level (red) in CSN5^null clones (arrows). (g) slimb^P1493, CSN5^null clones revealed by the absence of GFP (green) in wing discs show Ci¹⁵⁵ (red) accumulation in the A compartment (arrow) except when located adjacent to the A/P boundary (arrowhead). (h) The Ci-3P mutant protein (red), carrying mutations in three PKA phosphorylation sites, is ectopically expressed in CSN4^null mosaic wing pouches under the control of ms1096-GAL4. There is no difference in Ci-3P expression in wild-type and GFP-negative CSN4^null cells (arrow). Scale bars in all panels represent 50 μm.