FIGURE 6.

The α4s-containing CP is the predominant form of CP in mature sperm. A, extracts of adult mouse sperm were fractionated by glycerol gradient centrifugation. The chymotrypsin-like activity of the resultant fractions was measured in the presence of 0.025% SDS (top panel). Arrows indicate the peak locations of the 20 S CP and the 26 S proteasome. Each fraction was subjected to immunoblotting with the indicated antibodies (bottom panel). B, extracts of adult mouse testes and sperm were subjected to immunoblotting with the indicated antibodies. C, equal amounts of mouse sperm extracts (input) were immunoprecipitated with either anti-α6 antibody or anti-α4s antibody. The resultant unbound fractions as well as the input were subjected to immunoblotting with the indicated antibodies. D, immunoprecipitates (IP) with anti-α6 antibody were serially diluted (20, 40, 60, 80, and 100%) and subjected to immunoblotting for α6. Each band was quantified by densitometry and used to generate a standard curve for estimation of the amount of α6. The band of α6 in the immunoprecipitate with anti-α4s antibody was quantified following estimation of its amount by interpolation on the standard curve (dotted line). E, mouse sperm was incubated with dimethyl sulfoxide (DMSO) or 10 μm epoxomicin for 7 h and lysed for immunoblot analysis with antibodies against ubiquitin (Ub) and Rpt6. Data in are representative of at least three independent experiments.