Protein phosphatase inhibitor cocktail 3

HeLa cytosol was isolated after swelling suspension cells in hypotonic buffer (0.75 Mg(OAc)₂, 0.15 mM EDTA, 1 mM PMSF, 0.01 mg/ml leupeptin, 20 mM pepstatin A, 3 mM DTT), followed by dounce homogenization and clarification (16,000×g, 20 min, 4°C). TB (1/10 volume, 10× at 20 mM hepes pH 7.3, 2 mM Mg(OAc)₂, 110 mM K(OAc), 1 mM EGTA) was added before storage at −80°C. Whole cell lysates were prepared similarly, by the addition of WCLB (50mM Tris pH7.4, 50 mM NaF, 5mM Na₄O₇P₂, 1 mM EDTA, 1mM EGTA, 250mM mannitol, 1% triton ×100; 1mM DTT, 1mM PMSF, 20 mM pepstatin A, and 1× protein phosphatase inhibitor cocktail 3, Sigma Aldrich) to cells, followed by sonication (2×, 30 sec, 4°C) and clarification (16,000×g, 10 min). The materials were aliquoted and snap-frozen before storage at −80°C. Nuclei were isolated from suspension cells following treatment with digitonin and clarification to remove cytosol (44 (link)). Briefly, the cells were collected, washed (2×, PBS), then incubated (10 min, 4°C) in RSB (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2.5 mM MgCl) with digitonin (40ug/ml, Sigma Aldrich). The samples were clarified (2000×g, 8 min), and the pellets were rinsed with RSB, then TB (plus 1 mM DTT, 3×). Quantitation was with a hemocytometer after staining with tryphan blue.

HeLa nuclei (10⁶ cell equivalents) and fractionated cytosol (3×10⁵ cell equivalents) were combined with γ-³²P-ATP (10 μCi). Recombinant GST or GST-L_E (2 μg), were added. Some samples were supplemented with okadaic acid (250 nM). Reactions were incubated at 37°C for 45 min before the addition of RIPA (300 μl/sample, 50 mM Tris, pH 7.4, 150 mM NaCl, 0.1 % SDS, 1% Triton ×100, 0.5% Na deoxycholate, 1mM PMSF, 20 mM pepstatin A, and protein phosphatase inhibitor cocktail 3, Sigma Aldrich) and sonication. Protein G-conjugated beads (10 μl, G.E. Lifesciences) were added. Incubation was for 1 hr before the beads were removed by centrifugation. Fresh protein-G beads conjugated to α-Nup50 or α-Nup62 (saturated) were incubated with agitation (4°C, 3 hr) and then collected. After extensive washing (6×, PBS with 0.02% triton), samples were denatured with SDS buffer, boiled and fractionated by SDS-PAGE. Protein bands were detected by silver-staining and autoradiography, with densitometry performed using a Typhoon imager (GE Healthcare).