Phosphatase inhibitor cocktail tablets phosstop

parental PC12 cells and/or TrkB-PC12 were harvested from T-75 flasks, centrifuged and subjected to the needle aspiration procedure described above. Cells were seeded on 100 × 20 mm Corning BioCoat collagen I-coated dishes (Fisher Sci #356450) in complete growth medium at a density of 1 × 10⁶ cells/ml (10 ml per dish) and grown for one day at 37°C in 5% CO₂. Before stimulation with ligands, cells were starved overnight in RPMI-1640 medium containing L-glutamine, HEPES, sodium pyruvate and penicillin-streptomycin. PC12 or TrkB-PC12 cells transiently transfected with siRNA p75^NTR or control siRNA for 72 hours were left unstimulated or were stimulated with 50 ng/ml BDNF (R&D Systems #248-BD reconstituted in PBS with 1 mg/ml BSA) or 50 ng/ml rat NGF from R&D Systems (#556-NG-100) for the time intervals shown at 37°C in humidified 5% CO₂. Cells were lysed using ice-cold Cell Lysis Buffer (CST #9803) supplemented with Phosphatase Inhibitor Cocktail Tablets PhosSTOP (Roche #04906837001) and Protease Inhibitor Cocktail Tablets Complete (Roche #11697498001). Cells were scraped from the surface, harvested, vortexed, incubated for 30 min on ice, and then centrifuged at 10,000 × g for 10 min at 4°C. Aliquots of supernatant were dissolved in NuPAGE LDS sample buffer (ThermoFisher Sci #NP0007) supplemented with 50 mM DTT and heated for 7 min at 75°C.