sex. The DGE data showed that in C. capitata the six SR encoding genes are transcribed and most likely translated also in the female adult flies. Based on our study we can only speculate that 1) the 6 G5555 site Ceratitis SR proteins are transcribed and expressed in both sexes to perform basal alternative splicing regulations, related to their deep evolutionary metazoan conservation and 2) that the malespecific mAb104-detected SR phosphorylated proteins could have additional functions, possibly related to maleness, and to the control of sex-specific or sex-biased gene splicing. As Ceratitis, Musca and Drosophila show a conservation of tra and dsx sex-specific splicing regulation, but only Ceratitis show male-specific SR phoshorylated proteins, we propose that these splicing factors are involved in regulatory events either upstream or downstream to the tra>dsx regulatory PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19796427 module. We think that the observation of male-specific phosphorylated SR proteins in Ceratitis male flies is a preliminary necessary premise to provoke interest and to start further in silico and functional analysis also in other Tephritidae. Future experiments including SR proteins extraction from dissected XY fly body parts and from sexed embryos/ larvae will help to understand where and when the male-specific SR phosphorylation pattern is present, although we presently think that these SR antigens are present in most of the tissues of the adult male fly. Immunoprecipitation and sequencing will help to find further confirmation and information about identity of the SR antigens and related potential isoforms. Materials and methods Immunoblotting 3-4 gr of flies were used for each protein sample and the final Mg++ pellet was resuspended in solution and entirely loaded in one gel lane. During the purification procedure an aliquot from each of three different steps was saved for gel electrophoresis analyses. Tissues were ground to a fine powder in liquid nitrogen, using a mortar and pestle, and transferred to 30 ml of the isolation buffer, 5 mM p-glycerophosphate, 0.2 mM PMSF, and 2 g/ml of aprotinin). The samples were sonicated and centrifuged to eliminate debris; from the supernatant a first aliquot of total proteins was saved from each sample for gel analysis. The supernatant was used for a 65% and then a 90% ammonium sulfate precipitations. A second aliquot from the supernatant of the centrifuged 90% ammonium sulfate precipitation was saved for analyses. The pellets were resuspended in 200 microl of dialysis buffer, dialyzed against three changes of 300 ml of dialysis buffer over the course of 9 hr. The dialysate was recovered and stored at -80C. The dialysate samples were thawed and centrifuged for 15 min at 13,000g. Supernatants were transferred to clean tubes, and MgCl2 was added to 20 mM. After a 1-hr incubation on ice, tubes were centrifuged at 13,000g for 30 min. After removal of the supernatants, from which samples were saved for gel analyses, the pellets were washed with 200 microliters of 20 mM MgCl 2 dialysis buffer and resuspended in 20 micro1iters of 5% glycerol buffer D. Sample buffer was added to the various samples obtained from the purification procedure and the proteins were separated by SDS-10% PAGE. Similarly to the observations of Zahler et al., the complexity of the protein samples throughout the 4 purification steps, was gradually reduced from step 1 to step 4, leading approximately to Mg++ pellets containing 1-2 dozens prominent polypeptides visible by blue
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