Posts Tagged ‘Ribonucleic Acid

Males homozygous for the tw8 haplotype are sterile. As those studies describe, testes from tw8/tw8 males have many degenerating seminiferous tubules that are devoid of germ cells. Other tubules undergo grossly normal spermatogenesis; however, many spermatids have abnormally shaped heads and dumbbell-shaped nuclei. Some tubules also seem to be less organized than in wild type, having […]

Confocal micrographs of SPNR staining in hop/hop testes show that, as in wild type, SPNR forms as a caplike structure around the caudal end of the nucleus in step 9 spermatids, entirely coincident with tubulin staining (arrowhead, Fig. 3L). SPNR staining continued to overlap with tubulin staining as the spermatids formed long, abnormal manchette microtubules […]

SPNR was localized by immunocytochemistry (Fig. 1) and by immunofluorescence (Fig. 3) to Carnoy’s-fixed sections of testis from azh/azh males. Confocal microscopy of azh/azh sections labeled with anti-SPNR (Fig. 3, A and D) and anti-tubulin antibodies (Fig. 3, B and E) showed that as in the wild-type testis (Fig. 2), SPNR is coincident with tubulin. […]

Immunocytochemical localization of the SPNR protein in wild-type, azh/azh, hop/hop, tw8/tw8, and tw2./tw2 mutant testes is shown in Figure 1. Confocal micrographs of SPNR (fluorescein isothiocyanate [FITC], green) and tubulin (Texas red) immunofluorescence in wild-type and mutant testes are shown in Figures 2-4. Spermatogenesis in the mouse has been divided into 12 developmental stages based […]

Western Analysis Protein extracts were mixed with Laemmli buffer, boiled for 5 min, and electrophoresed on 8% SDS-PAGE gels. The gels were blotted to nitrocellulose overnight at 4°C using an electroblotter run with water-cooling at 200 mA in a Tris-glycine buffer (25 mM Tris, 192 mM glycine, 20% v:v methanol, pH 8.3). The blots were […]

To assess binding to mouse testis microtubules, microtubule pellets from the first round of purification (described above) were resuspended in 120 ^l His-SPNR E. coli extract. The mixture was incubated at 37°C for 15 min, and microtubules were pelleted by spinning at 30 000 X g for 20 min at 37°C. The pellets were resuspended […]

The microtubule pellet was rinsed in MT buffer and resuspended in 125 MT buffer supplemented with taxol (20 ^m final concentration), GTP (1 mM final concentration), and protease inhibitors (as above). The tube was placed at 37°C for 15 min, and microtubules were pelleted as described above. The pellet was resuspended in MT buffer and […]

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