Cavener, Penn State University); rabbit anti-AP (Serotec, used at 1:600); rabbit-anti-RFP (dsRed) (Rockland, used at 1:1,000); rabbit anti-GFP (Invitrogen, used at 1:1,000); Rhodamine-conjugated phalloidin (Invitrogen, used at 1:2,000); AlexaFluor 488 anti-mouse and AlexaFluor 568 anti-rabbit (Invitrogen, used at 1:1,000); and HRP-conjugated goat-anti-mouse and goat anti-rabbit (Jackson ImmunoResearch, used at 1:150). The Sas cDNA construct was made using a full-length cDNA clone for the 1693 aa protein. This was inserted into pUAST-attB, and site-specific X and second chromosome transgenics were made by Rainbow Genetics. Expression

of RPTP-AP proteins using baculovirus was described by Fox and Zinn (2005). Sas-Fc was made by inserting the entire Alisertib nmr XC domain sequence of Sas into an S2 expression vector containing the human Fc sequence with a His tag (Wojtowicz et al., 2007). Then, the entire CH5424802 concentration Sas-Fc coding region, minus the signal sequence, was amplified by PCR from this plasmid and inserted into a baculovirus vector, pAcGP67A. Sas-Fc was purified

from supernatants of cells infected with the virus derived from this vector, using Ni-NTA agarose. We PCR-amplified exons of the sas15 gene from sas15 homozygote larvae and sequenced multiple clones from multiple amplifications to ensure that observed changes were due to mutation and not to PCR errors. We observed changes from wild-type as follows: exon 2, position 3,530, noncoding; exon 2, position 3,590, noncoding (5 bp deletion); exon 2, position 3,784, missense; exon 6, position 17,890, nonsense (stop codon mutation at aa 642); exon 6, position 18,024, missense; exon 9, position 20,000, missense. Each well of Nunc Immunosorb 96-well plates was incubated overnight at 4°C with 50 μl (3 μg/ml antibody) of unpurified ascites fluid containing the IgG2A anti-AP mAb 8B6 (Sigma)

in 1× PBS, pH 7.4. Wells were washed five times for 1–3 min at MycoClean Mycoplasma Removal Kit room temperature with 150 μl 1× PBS, pH 7.4 + 0.05% Tween-20 (PBST). Wells were incubated for 1–2 hr at room temperature with 150 μl 1% casein in 1× PBS, pH 7.4, on a rocking platform. The 1% casein block was removed. This was followed by the addition of 20 μl Fc fusion protein (Sas-Fc [5 ng/μl], Unc5-Fc [5 ng/μl] or FasII-Fc [5 ng/μl]) and 20 μl AP fusion protein (10D-AP [8.5 ng/μl], Lar-AP [8.5 ng/μl], 69D-AP [8.5ng/μl], or blank culture medium), for a total volume of 40 μl. The AP fusion protein dilutions also contained HRP-conjugated mouse anti-human IgG1 (2 μg/ml; Serotec). Plates were covered and incubated overnight at room temperature protected from light. The next day, wells were washed five times for 1–3 min at room temperature with 150 μl PBST. 1-Step Ultra TMB-ELISA HRP substrate (100 μl; Pierce catalog 34028) equilibrated to room temperature was added and plates were incubated for 1 hr at room temperature. Absorbance at both 370 nm and 652 nm wavelengths was measured using an ND-1000 spectrophotometer.