Fig. 1.

The crb locus encodes four different protein isoforms. (A) Four crb mRNA variants are the result of alternative splicing. Exons 1-3, 6 and 8-16 are shared. Exon 4 is specific for crb-RB and crb-RD, exon 5 is only found in crb-RC and exon 7 only in crb-RD. Grey boxes indicate 5′ and 3′ UTRs. (Adapted from Flybase.) (B) The four predicted Crb isoforms. Green rectangle: EGF-like repeat (numbering based on Crb_A), pink hexagon: repeat with similarity to the globular domain of laminin A, TM: transmembrane domain. The four isoforms have the same N-terminus, including EGF-like repeat #1-6, and share the C-terminus from EGF-like repeat #8 onwards, except for EGF-like repeat #11, which carries a 12 amino acid insertion in Crb_D. Isoforms B and D lack EGF-like repeat #7, isoform C carries an additional EGF-like repeat (#7a). The horizontal bars indicate the regions used as epitopes for immunization. Antibodies indicated on top detect all isoforms. α-Crb_C is specific for isoform C, α-Crb_B/D is specific for isoforms B and D. The blue line in isoform C indicates the isoform-specific insertion (see also Fig. S1). (The current version of SMART used does not suggest an EGF-like repeat between #8 and #9, nor an additional laminin A-like repeat N-terminal to EGF-like repeat #1 as shown in some previous publications.) (C) Western blot to demonstrate the specificity of α-Crb_C antibody. Crb_A and Crb_C were overexpressed in Drosophila S2R⁺ cells and cell extracts were probed with either α-Crb_intra, which detects both isoforms, or α-Crb_C. Upon strong overexpression, α-Crb_C detects two bands, which presumably represents the non-glycosylated precursor and the mature glycoprotein, as upon deglycosylation only a single band is detected (shown in D). (D) Crb_A and Crb_C were overexpressed in Drosophila S2R⁺ cells, cell extracts were deglycosylated and probed with α-Crb2.8. Crb_C runs slightly slower than Crb_A, independent of whether it is glycosylated or not.