Fig. 6. Western blots with epitope tagged Grk protein and GST pull-downs. (A) Grk tagged C-terminally with five myc tags (Grk5myc) under endogenous control. Only the C-terminal cleavage residue but not a full-length precursor can be detected using the 9E10 anti-myc monoclonal. No differences are visible between homozygous cni^AR55 mutant ovaries and heterozygous sibling controls. (B) Grk5myc overexpressed in wild-type (+) and homozygous cni^AR55 (cni) ovaries using the TubGal4VP16 driver. The 9E10 anti-myc monoclonal reveals two bands. Although the lower band corresponds in size to the expected C-terminal cleavage residue, the upper band, corresponding to an uncleaved precursor, is larger than predicted from the protein sequence, indicative of glycosylation. In addition to the precursor band, the 1D12 anti-Grk monoclonal reveals a shorter band corresponding in size to the released N-terminal growth factor fragment in the mutant but not the wild-type lane. (C) A protein species corresponding in size to the uncleaved Grk precursor accumulates in lysates expressing a Grk5myc construct carrying the grk^DC point mutation from the endogenous promotor (GrkDC5myc). This does not occur in ovaries expressing the functional Grk5myc version. Smearing of the GrkDC5myc band into several high molecular weight species indicative of Golgi glycosylation only occurs in the presence of Cni. (D) The N-terminal growth factor fragment accumulating in cni mutant ovaries upon Grk overexpression is sensitive to EndoH and PNGaseF, indicating ER-type high Mannose glycosylation. (E) Grk and Cni interact directly. MBP fusion proteins with lacZ or the N-terminal 57 amino acids of Cni or Cnir were pulled down using GST or GST-Grk (GST fused to Grk amino acids 179-245) prebound to beads. Probing the pellets with anti-MBP antibody reveals a specific interaction between the juxtamembrane domain of Grk and the Cni N terminus.