Halt phosphatase inhibitor cocktail

Phosphorylated Fli-1 was detected as described previously (28 (link)). For the detection of acetylated Fli-1, cells were lysed in Pierce IP lysis buffer with the Halt phosphatase inhibitor cocktail (Thermo Scientific Pierce). Cell lysates were precleared with protein G–Sepharose (GE Healthcare) and then were incubated with monoclonal mouse anti–Fli-1 antibody (BD Biosciences) and protein G–Sepharose beads overnight at 4°C. The immunoprecipitated proteins were washed with Pierce buffer. Agarose-bound proteins were extracted by incubation in sample buffer at 95°C. The sample was then assessed by immunoblotting with anti–acetylated lysine antibody (Cell Signaling Technology). The membrane was stripped and reprobed with rabbit polyclonal anti–Fli-1 antibody (Santa Cruz Biotechnology) (29 (link)).

Total Tau was isolated from spinal cord or CC lysates using the Crosslink Immunoprecipitation Kit (Pierce). Tissues were homogenized in 10 volumes of cold Thermo IP Lysis Buffer supplemented with 3 μM trichostatin A (pyridine-3-carboxylic acid amide, Sigma), 10 mM niacinamide (Sigma) and Halt Protease and Phosphatase Inhibitor Cocktail (Fisher). Lysates were then sonicated on ice twice for 5 minutes with a sonicator at 40% amplitude and spun at 10,000×g for 10 minutes at 4 °C. Supernatants were collected and protein concentration was measured using the Bicinchoninic (BCA) Protein Assay Kit (Pierce). Lysate volumes containing 1 mg of total proteins were first pre-cleared with 40 μL of Control Agarose Resin for 1 hour at 4° C and then were incubated with 30 μg of TAU-5 mAb crosslinked to Protein A/G Plus Agarose beads for 10 hours at 4 °C, rotating. Beads were washed 4 times with IP lysis buffer and once with TBS before eluting the bound proteins with 50 μL of Pierce Elution Buffer. For in-solution trypsin digestion before mass spectrometry, immunoprecipitated samples were neutralized with 1 M Tris, pH 9.6, supplemented with 3 μM trichostatin A, 10 mM niacinamide and Halt Phosphatase Inhibitor Cocktail (Fisher).

For preparation of cell extract, cells at 90% confluency were lysed in buffer containing 50 mM Tris (pH 7.5), 0.1% NP-40, 100 mM NaCl, 1 mM MgCl2, 5 mM EDTA, protease inhibitor cocktail (Fisher) and Halt phosphatase inhibitor cocktail (Fisher) on ice for 30 min. Cell lysates were sonicated for 10 pulses at level 1 with 10% output 3 times. The lysates were incubated on ice for another 10min and then centrifuged at 17,000g for 20 min at 4 °C. For preparation of mouse brain extract, brains were quickly dissected and dounce-homogenized in the above lysis buffer. The lysates were incubated on ice for 30 min and then centrifuged at 17,000g for 30 min at 4 °C. Concentrations of extracts were determined by a BCA protein assay (Pierce).