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Characterization of D1Pas1, a mouse autosomal homologue of the human AZFa region DBY, as a nuclear protein in spermatogenic cells Donna R Session, M.D., Grace S Lee, B.A., Debra J Wolgemuth, Ph.D. Fertility and Sterility Volume 76, Issue 4, Pages (October 2001) DOI: /S (01)
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FIGURE 1 In vitro transcription/translation of D1Pas1. The 35S-labeled in vitro translation product was run on a 10% SDS-PAGE and the autoradiograph exposed overnight. A single band of the predicted size is detected in the samples of the protein derived from the 3.2-kb D1Pas1 cDNA (3). A band was not detected in the control lane, which included linearized vector with an untranslatable cDNA fragment. The relative molecular weight of the protein is indicated. Session. D1Pas1 and spermatogenesis. Fertil Steril 2001. Fertility and Sterility , DOI: ( /S (01) )
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FIGURE 2 Detection of D1Pas1 native and fusion protein. Lysates of the fusion protein (F) and total testis (T) of adult Swiss Webster mice were immunoblotted with 1 μg/mL of affinity-purified antibody. The molecular weights of the markers are indicated, and the entire blot is represented. Sixty micrograms of protein was loaded per lane. Session. D1Pas1 and spermatogenesis. Fertil Steril 2001. Fertility and Sterility , DOI: ( /S (01) )
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FIGURE 3 Subcellular expression of D1Pas1 in the testis. The relative molecular weights of the proteins are indicated. (A), Detection of D1Pas1 protein in separated testicular cells. Testicular cells were separated into purified pools of pachytene spermatocytes (P), round spermatids (RS), and cytoplasmic fragments and residual bodies (CF/RB). Lysates from the pooled cells were analyzed for the presence of D1Pas1 protein by immunoblotting with 1 μg/mL of purified antibody. Two hundred micrograms of total protein was loaded per lane. (B), Detection of D1Pas1 protein in a testicular cell fractionation. Sixty micrograms of protein was loaded per lane. Immunoblotting was performed using a 1:1,000 dilution of whole serum. Lane N = nuclear fraction; T = total testis; C = cytoplasmic fraction. Session. D1Pas1 and spermatogenesis. Fertil Steril 2001. Fertility and Sterility , DOI: ( /S (01) )
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FIGURE 4 Immunohistochemical localization of D1Pas1 in the testis. Immunohistochemistry was performed on paraffin-embedded Swiss Webster testis sections using a 1:500 dilution of whole serum on adult testis (A), day 17 testis (B) at ×100 magnification, and preimmune serum on day 17 testis (C) at ×40 magnification; and a 1:500 dilution of whole serum on adult testis (D) and day 17 (E) testis at ×40 magnification. The sections were stained brown with diaminobenzidine and counterstained with hematoxylin, which stains nuclei blue. Session. D1Pas1 and spermatogenesis. Fertil Steril 2001. Fertility and Sterility , DOI: ( /S (01) )
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FIGURE 5 Amino acid alignment of mouse and human genes. Amino acids identical in D1Pas1, DBX, and DBY are shown in upper-case letters. Dots indicate sequence breaks that are included to give optimal alignments. Shaded boxes correspond to the consensus motifs I-IX in proteins identified as DEAD box RNA helicases (27, 28). Session. D1Pas1 and spermatogenesis. Fertil Steril 2001. Fertility and Sterility , DOI: ( /S (01) )
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