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Figure S1. Loading control, related to Fig. 1F. SDS-PAGE corresponding to the in vitro kinase assay investigating the phosphorylation of the carboxy-terminal region of Spen encoded by cDNA clone SD17134. Hipk shows a strong tendency towards degradation, thereby partially superimposing the bands of potential substrate proteins. Marker used: ‘Page Ruler Unstained Protein Ladder’ (Fermentas).

Figure S2. Loading control, related to Fig. 3. SDS-PAGEs corresponding to the in vitro kinase assays investigating the phosphorylation of the cloned Nito fragments. Hipk shows a strong tendency towards degradation, thereby partially superimposing the bands of potential substrate proteins. Markers used: ‘Page Ruler Unstained Protein Ladder’ and ‘Unstained Protein Molecular Weight Marker’ (Fermentas). A gel referring to the analysis of the first nito constructs nitoA, nitoB and nitoC (Fig. 3B). B gel referring to the analysis of the subconstructs of nitoA (Fig. 3E). C gel referring to the analysis of the subconstructs of nitoC (Fig. 3F).

Figure S3. Loading control, related to Fig. 4. SDS-PAGEs corresponding to the in vitro kinase assays investigating the phosphorylation of the mutated Nito fragments. Hipk shows a strong tendency towards degradation, thereby partially superimposing the bands of potential substrate proteins. Markers used: ‘Page Ruler Unstained Protein Ladder’ and ‘Unstained Protein Molecular Weight Marker’ (Fermentas). A gel referring to the analysis of the mutated subconstructs of nitoA (Fig. 4B). B gel referring to the analysis of the mutated subconstructs of nitoC (Fig. 4B). C gel referring to the controls used for the mutational analysis (Fig. 4C).

Figure S4. Technical controls for bimolecular fluorescence complementation (BiFC) analysis of in vivo interactions between Hipk and Nito, related to Fig. 5. Third instar wing imaginal discs expressing NYFP-Hipk (A–C), Gro-CYFP (D–F) or Nito-CYFP (G–I), respectively. Each construct is expressed in the pattern of en-Gal4 (A,D,G), ptc-Gal4 (B,E,H) and MS1096-Gal4 (C,F,I). All discs are oriented anterior to the left and dorsal side up. Immunostaining was done using an anti-GFP-antibody providing a technical control against false-positive signals. Brightness of the images had to be artificially increased in order to properly visualise the discs, thereby leading to a slight baseline staining in some of the constructs. However, the signal strength of this staining is clearly below the strength of the signals based on complementation of the split YFP fragments due to protein-protein interactions, making them clearly distinguishable from each other.

Figure S5. Nito and Hipk do not exert an influence on Senseless expression on their own. Third instar wing imaginal discs, oriented anterior to the left and dorsal side up. Nito gene dose was reduced (A) and hipk gene dose was increased (B) independent from each other using a pct-Gal4 driver line, respectively. Neither of the expressed constructs led to significant alterations in the expression of the wingless target Senseless.

Appendix S1. Supplemental experimental procedures.

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