Purification and Proteinase K Digestion of Spermatogenic Cells

Spermatogenic cells were purified by the above method and subjected to in situ proteinase K digestion^{41 (link)}. In brief, after two rinses with ice-cold PBS, one lot was incubated in 4 µg/ml proteinase K (EO0491, Thermo Scientific) in KHM buffer (110 mM KOAc, 20 mM Hepes, pH 7.4 and 2 mM MgCl₂) for 45 min at room temperature. The second lot was permeabilized with 24 µM of ice-cold digitonin in KHM for 15 min followed by 4 µg/ml proteinase K digestion in KHM for 45 min at room temperature. The third lot was incubated with 4 µg/ml proteinase K in KHM containing 0.5% Triton X-100 for 45 min. Subsequently, PMSF was added to all lots to a final concentration of 40 μg/ml. Cells were then washed in KHM and lysed in 0.4% SDS, 2% Triton X-100, 400 mM NaCl, 50 mM Tris–HCl, pH 7.4, 40 μg/ml PMSF, 1 mM dithiothreitol, and protease inhibitors (04693132001, Roche) by passing through a 22-gauge needle before centrifugation for 10 min at 16,000 × g. Proteins were then probed with specific antibodies by western blotting.