DAF-25/Ankmy2 Functions in Ciliary cGMP Signaling Figure 6. Ankmy2 and GC1 can be co-precipitated in HEK293 cells. In (A), detergent-solubilized extracts of HEK293 cells co-expressing Ankmy2-1D4 and either GC1 of ABCA4 were immununoprecipitated on a Rho 1D4-Sepharose matrix and the bound protein was analyzed on Western blots labeled with Rho 1D4 for detection of Ankmy2 and antibodies to GC1 or ABCA4 to detect co-precipitating proteins. Precipitation of 1D4-tagged Ankmy2 with 1D4 antibody also pulls down GC1 (retinal guanylyl cyclase), but not ABCA4 (retinal expressed ATP-binding Cassette, sub-family A, member 4). This indicates that GC1 forms a protein complex with Ankmy2, implying conservation of the functional interaction between DAF-11 and DAF-25. Lane 1 indicates the input proteins (whole cell lysate) and lane 2 indicates elution from immunoaffinity matrix. In (B) detergent-solubilized extracts of HEK293 cells expressing only Ankmy2-1D4 or GC1 were immunoprecipitated on a Rho 1D4 immunoaffinity matrix and analyzed on Western blots labeled with an anti-GC1 antibody or Rho 1D4 antibody. Lane 1: Input; lane 2: bound protein. The presence of Ankmy2-1D4 but not GC1 in the bound fractions indicates that GC1 does not nonspecifically interact with the Rho 1D4 immunoaffinity matrix. In (C), HEK293 cells co- expressing Ankmy2-1D4 and GC1 were co-immunoprecipitated on a Rho 1D4 immunoaffinity matrix in the absence or the presence of excess competing 1D4 peptide. Both Ankmy2-1D4 and GC1 bound in the absence of peptide. In the presence of the 1D4 peptide less than 10% of the Ankmy2 bound. doi:10.1371/journal.pgen.1001199.g006 described [30]. ArrayCGH was done as previously described [47] for the leftmost 2.4 Mbp of Chromosome I with 50 base probes spaced every four base pairs. Epistasis analysis was performed by crossing daf-25(m362) into daf-12(m20), daf-16(m26), daf-3(mgDf90), daf-10(e1387) and daf- 6(e1377). Once the double mutants were isolated, the dauer phenotype was assayed to determine if daf-25 was suppressed fully (no constitutive dauer larvae formed at 25uC), partially (fewer dauer larvae than daf-25(m362) control) or no suppression. Treatment with cGMP was performed as previously described [12] with 5 mM 8-bromo-cGMP (Sigma). Neuronal dye-filling was assayed by incubating a mixed-stage population of each genotype in Vibrant DiI (Molecular Probes) 1000-fold diluted in M9 buffer for one hour followed by washing in M9 and one hour destaining on plates. Chemotaxis assays were performed synchronized day-1 adults as previously described with the volatile attractants trimethyl-thiazole, pyrazine, benzaldehye and isoamyl alcohol [48]. The DAF-25::GFP construct was created by inserting the 2.0 kb promoter region 59 of the AUG followed by daf-25 cDNA the into the pPD95.77 vector (gift from Dr. Andrew Fire). After microinjection into N2 adults [49] with 10 ug/ml of pRF4 (contains rol-6(su1006)), and 90 ug/ml of DAF-25::GFP plasmid (described above), transgenics lines were established based on the roller phenotype. The extra-chromosomal array mEX179(pdaf- 25::DAF-25::GFP, rol-6(su1006)) was crossed into daf-25(m362) and rescue of the Daf-c phenotype was detected by normal non- dauer development in the F3 progeny grown at 25uC. GFP fluorescence was visualized on a Zeiss Axioskop with a Qimaging Retiga 2000R camera. PLoS Genetics | www.plosgenetics.org 9 November 2010 | Volume 6 | Issue 11 | e1001199