Phosphatase inhibitor

HLFs were grown to approximately 70–80% confluence and cultured with serum-free MEM for 18 h before collection. Cells were collected in PBS and centrifuged at 1500 rpm at 4 °C for 5 min. The cell pellets were lysed (50 mM Tris PH 8; 0.5% Triton X100; 450 mM NaCl; Protease Inhibitor Cocktail; Phosphatase Inhibitor (Roche, Laval, QC, Canada), incubated for 15 min on ice, and then centrifuged at 10,000 rpm, 4 °C for 15 min. The extracts were transferred into as new tube and a buffer containing 50 mM Tris pH 8; 0.5% Triton X100; 10% glycerol; Protease Inhibitor Cocktail; Phosphatase Inhibitor (Roche, Laval, QC, Canada) [35 (link),36 (link)] was added. Protein concentration was measured by the BCA Protein Assay Kit. Thirty-five μL of protein G Sepharose^TM 4 fast glow beads (GE Healthcare, Mississauga, ON, Canada) were pre-coated with 3 μg of IgG (Cell Signaling Technologies, Whitby, ON, Canada) or 3 μg of anti-HuR (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies overnight on a rotator at 4 °C. Beads were washed three times with buffer (50 mM Tris PH 8; 0.5% Triton X100; 150 mM NaCl) and incubated with cell extracts for 2 h a 4 °C. Beads were washed three times to wash out unbound materials. RNA was then extracted, reverse transcribed and analyzed by qPCR (RT-qPCR), as described above. RNA expression was normalized to S9 mRNA bound in a non-specific manner to IgG.

Testes were removed from male C57BL/6J mice and suspended in extraction buffer (20 mM Tris-HCl (pH7.5), 50 mM KCl, 0.4 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mM β-mercaptoethanol) supplemented with cOmplete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). After homogenization, the cell extract was centrifuged at 50,000 × g for 30 min at 4 °C and the supernatant was isolated. The extract was supplemented with Dynabeads protein A (Thermo Fisher Scientific) conjugated with 80 μg of anti- MEILB2 antibody or control IgG as the negative control and incubated for 6 h at 4 °C. The beads were washed with high-salt buffer (20 mM HEPES (pH 7.0), 400 mM KCl, 5 mM MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mM β-mercaptoethanol) supplemented with cOmplete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). The samples were eluted with 0.1 M glycine (pH 2.5).

Transfected B16-F1 cells were suspended in extraction buffer (20 mM Tris-HCl (pH7.5), 50 mM KCl, 0.4 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mM β-mercaptoethanol) supplemented with complete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). After sonication, the cell extract was centrifuged at 15,000 × g for 30 min at 4 °C and the supernatant was isolated. The supernatant was then incubated with GFP-trap®_MA beads (ChromoTek) for 2 h at 4 °C on a rotating wheel. Beads were washed with high-salt buffer (20 mM HEPES (pH 7.0), 400 mM KCl, 5 mM MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mM β-mercaptoethanol) supplemented with complete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). The samples were eluted with sodium dodecyl sulfate loading buffer at 95 °C for 5 min.

Mixed-stage worms were collected and suspended in IP buffer (20 mM HEPES (pH 7.0), 200 mM KCl, 5 mM MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mM β-mercaptoethanol) supplemented with cOmplete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). After sonication, the cell extract was centrifuged at 50,000 × g for 30 min at 4°C and the supernatant was isolated. The extract was supplemented with Dynabeads protein A (Thermo Fisher Scientific) conjugated with 80 μg of antibodies or IgG as the negative control and incubated for 6 hr at 4°C. The beads were washed with high-salt buffer (20 mM HEPES (pH 7.0), 400 mM KCl, 5 mM MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mM β-mercaptoethanol) supplemented with cOmplete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). The samples were eluted with 0.1 M glycine (pH 2.5).

Transfected B16-F1 cells were suspended in extraction buffer (20 mm Tris-HCl (pH 7.5), 50 mm KCl, 0.4 mm EDTA, 5 mm MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mm β-mercaptoethanol) supplemented with cOmplete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). After sonication, the cell extract was centrifuged at 15,000 × g for 30 min at 4°C and the supernatant was isolated. The supernatant was then incubated with GFP-trap Magnetic Agarose (Chromotek), Myc-trap Magnetic Agarose (Chromotek), or Anti-FLAG M2 Magnetic Beads (Sigma) for 2 h at 4°C on a rotating wheel. The beads were washed with high-salt buffer (20 mm HEPES (pH 7.0), 400 mm KCl, 5 mm MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mm β-mercaptoethanol) supplemented with cOmplete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). The samples were eluted with SDS loading buffer at 95°C for 5 min.

Testes were removed from male C57BL/6J mice and suspended in extraction buffer (20 mm Tris-HCl (pH 7.5), 50 mm KCl, 0.4 mm EDTA, 5 mm MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mm β-mercaptoethanol) supplemented with cOmplete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). After homogenization, the cell extract was centrifuged at 50,000 × g for 30 min at 4°C and the supernatant (chromatin extract) was isolated. The extract was supplemented with Dynabeads protein A (Thermo Fisher Scientific) conjugated with 80 μg of BRME1, MEILB2, or BRCA2 antibodies or control IgG and incubated for 6 h at 4°C. The beads were washed with high-salt buffer (20 mm HEPES (pH 7.0), 400 mm KCl, 5 mm MgCl₂, 10% glycerol, 0.1% Triton X-100, and 1 mm β-mercaptoethanol) supplemented with cOmplete Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). The samples were eluted with 0.1 m glycine (pH 2.5).

Total proteins of tissues and cells were extracted by RIPA buffer (Beyotime, P0013B) supplemented with 1% (v/v) 100 mM PMSF and phosphatase inhibitor (Roche, 4906837001). For immunoprecipitation, cells were lyses in non-ionic detergent RIPA buffer (Beyotime, P0013D) supplemented with 1% (v/v) 100 mM PMSF and phosphatase inhibitors (Roche, 4906837001). The Western blot and immunoprecipitation protocol is described in detail in the Supplementary Materials and Methods.