After addition, the buffer cells were sonicated for 30 s and centrifuged at 14 000 r.p.m. for 10 min and the supernatant was collected.
After resuspension in 1 ml lysis buffer (10 mM Tris (pH 8.0), 20% sucrose, 50 mM NaCl, 10 mM EDTA, 10 mg/ml lysozyme) and incubation at 37°C for 30 min followed by addition of 4 ml IP buffer, cells were sonicated on ice with 12 times 30 sec and 30 sec breaks at an UP 400 s Ultrasonic processor (Dr. Hielscher GmbH) with 100% power.
After being incubated with the label for 30 min, the cells were rinsed in ice-cold PBS containing 10 mM methionine and scraped gently from the filter in 0.2 mL buffer A. The cells were sonicated for 10 seconds and centrifuged for 5 minutes at 13,000 g in a refrigerated microfuge.
Cells were sonicated in buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) with 1% fresh stock cocktail of protease inhibitors (final concentrations: pepstatin A 50 μg/mL, chymostatin 50 μg/mL, leupeptin 50 μg/mL, and aprotinin 100 μg/mL).
Cells were sonicated in lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 0.5 mM EDTA, 5% glycerol, 0.1% Triton-X, 1 mM DTT and 1 mM protease inhibitor) and recombinant His6-tagged proteins purified using Ni2+-nitrilotriacetic acid His-Select resin (Sigma) and eluted with elution buffer (20 mM Tris, 200 mM NaCl and 200 mM imidazole).
Cells were sonicated in lysis buffer containing an EDTA-free complete protease inhibitor (Roche).
Cells were sonicated in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 9 mM sodium ortho-vanadate, protease inhibitor cocktail).
Cells were sonicated in a buffer solution 300 mM NaCl, 30 mM imidazole, 10 μg/mL DNAseI (Sigma-Aldrich, Milan, Italy), 0.19 mg/mL PMSF (Sigma-Aldrich, Milan, Italy) and 0.7 μg/mL pepstatine (Sigma-Aldrich, Milan, Italy) in 50 mM potassium phosphate buffer (pH 7.0), for 15 cycles of 30 seconds each on ice.
For α-syn blots of stable cell lines, cells were sonicated in lysis buffer (175 mM NaCl, 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, protease inhibitor cocktail (Roche Diagnostics)) and incubated with 1% Triton X-100 for 30 min.
Briefly, cells were sonicated in HCMF buffer containing 1% Triton, 0.1% SDS, 2 mM Calcium Chloride (CaCl2), 100 μg/mL phenylmethylsulfonyl fluoride (PMSF), and 1 μg/mL leupeptin at an intermediate setting (output ≅ 3) using a Branson Sonifier 250 (VWR Scientific, OH, USA).
KHYG-1 cells were sonicated in lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% (w/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 μg/ml leupeptin, and 2.5 μg/ml aprotinin) and incubated on ice for 30 minutes.
