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Rat Testis Motor Proteins Associated with Spermatid Translocation: MATERIALS AND METHODS(5)

MATERIALS AND METHODS(5)

Two equal aliquots of cell suspensions were taken and again washed in MES buffer. One pellet was resuspended in MES buffer, and the other pellet was suspended in an equal volume of gelsolin dialysate (0.4 mg/ml gelsolin in MES buffer). Samples were incubated for 1 h at room temperature with occasional gentle agitation. After incubation, small aliquots (2 ^l) of the MES control and gelsolin-treated cells were placed on polylysine-coated slides and stained with Oregon green-conjugated phalloidin. The slides were evaluated by fluorescence microscopy for the status of actin filaments in spermatid-associated Sertoli cell junction plaques. In this material, it was noted that the tails had separated from many of the spermatids. Remaining samples were centrifuged (4000 rpm for 2 min in Eppendorf centrifuge) to sediment out intact spermatids and spermatid heads, and the supernatants were frozen at —70°C in 10-^l aliquots. ampicillin antibiotic

Gel Electrophoresis and Immunoblots

Before electrophoresis, protein samples were diluted 1: 1 with treatment buffer, boiled for 2 min, and then loaded into wells. Proteins were separated on 10% polyacrylamide gels and transferred overnight to PVDf membranes. Blots were washed twice (10 min each wash) in TTBS (100 mM Tris-Cl pH 7.5, 0.9% NaCl, 0.1% Tween 20) and then blocked for 2 h in 10% nonfat dry milk plus 4% BSA in TTBS. The two 10-min washes were repeated, after which the blot was incubated for 1 h with freshly prepared primary antibodies (1:1000 dilution in TTBS) to cytoplasmic dynein and kinesin II. Mouse IgG controls at equivalent protein concentrations were run in parallel. Blots were washed (3 X 5 min, 3 X 10 min) and then treated with avidin-biotin-peroxidase complex (ABC) reagent (Vecta-stain ABC kit, Vector Labs) made up in a high-salt (0.5 M NaCL) TTBS solution for 30 min. Final washes were in TBST (3 X 5 min) and TBS (100 mM Tris-Cl pH 7.5, 0.9% NaCl) (3 X 10 min). Proteins were detected by chemilu-minescence using ECL reagent (Amersham, Piscataway, NJ). Molecular weights were approximated against bioti-nylated protein molecular weight markers (Vector Labs).

Immunoblots were scanned into digital format and manipulated, using Adobe Photoshop 4, without altering the integrity of the data.