Testis Lysate Phosphatase Assay

Total testis lysates were prepared as described above but phosphatase inhibitors were omitted. Each pair of testes from one juvenile male mouse was lysed in 50 µL 1x lysis buffer. After 1.5 h of incubation sample was centrifuged 16 000 g for 10 min. Then supernatant was diluted 2 times with phosphatase dilution buffer: 100 mM NaCl, 50 mM Tris-HCl pH 7.5 supplemented with protease inhibitors. Afterwards, lysate was split into 4 aliquots (35 µL each). All aliquots were supplemented with 10X NEBuffer for Protein MetalloPhosphatases (PMP) and 10X MnCl2 (NEB). The first aliquot (Untreated control) was supplemented with phosphatase inhibitors (PPase inhibitors): 0.5 mM sodium orthovanadate, phosphatase inhibitor cocktail 1 (Sigma, P2850) and phosphatase inhibitor cocktail 2 (Sigma, P5726). They were used at concentrations recommended by the manufacturers. After that 2x Laemmli buffer (with 10% b-Mercaptoethanol) was added and sample was boiled 95 ^°C for 7 min. The second (PPase inhibitors only) and third (PPase inhibitors + Lambda PPase) aliquots were also supplemented with PPase inhibitors. 2 µL of Lambda Protein Phosphatase (NEB, P0753S) was added to aliquots 3 and 4 (PPase only). Final volume of each sample was 50 µL. Aliquots 2-4 were incubated at 30 ^°C for 1.5 h. After incubation, 2xLaemmli buffer (with 10% b-Mercaptoethanol) was added 1:1 ratio and samples were incubated at 95 ^°C for 7 min.