Fig. 7. Post-translational modification of Knk depends on Mmy activity. (A) Western blot with protein extracts from wild-type and different mutant larvae labelled with a specific antibody against the extracellular cuticle protein Knk. In the wild type, Knk migrates as a single band (ExB and EndoH-), and EndoH treatment to cleave N-glycosylated sugar-residues generates smaller co-migrating Knk species (EndoH+). In mmy^IK63 and mmy^KG08617, several Knk proteins with different sizes are present. No Knk protein is detected in knk^14D79 mutants, and Knk protein migration is not affected in the remaining mutants tested. (B) Western blot with the same extracts as in A labelled with antisera against the membrane-attached Syntaxin1A (Syx1A), the transmembrane protein Tout-velu (Ttv) and cytosolic -Tubulin (Tub) as loading control. Ttv also migrates as a smaller protein after EndoH-treatment, and in mmy^IK63 and mmy^KG08617 mutant larvae differently sized Ttv proteins are present (stars). (ExB, extraction buffer PLC). (C-E) Co-labelling of stage 16 wild-type, knk mutant and mmy^IK63 mutant epidermis with anti-Knk (green) and anti-Fas3 (red). Knk localises to the apical plasma membrane in the wild type (C) and is absent in knk mutants (E). In the mmy^IK63 mutant epidermis, the amount of Knk is severely reduced in the apical membrane, and some signal is detected within the cell (D).