Complete protease inhibitor cocktail and phosphatase inhibitors

Proteins were collected from cultured cells and lysed with 10X Lysis Buffer (Cell Signaling) freshly supplemented with a complete protease inhibitor cocktail and phosphatase inhibitors (Sigma). Bradford assay was used to measure protein concentration. Proteins were separated by polyacrylamide gel (Bio-rad) electrophoresis and were transferred to a 0.2 µm nitrocellulose membrane (Bio-rad). The list of antibodies is described in Supplementary Table 3.

Crude tissue homogenates (20% w/v) were prepared in 100 mM Tris buffer (pH 7.0) containing 5% glycerol, 0.1% SDS, Complete Protease Inhibitor Cocktail and phosphatase inhibitors (P2850, P5726, Sigma-Aldrich), with the addition of 200 µM phenylmethyl-sulphonyl fluoride (PMSF; Sigma-Aldrich) and 157 µg/mL benzamidine hydrochloride (Serva, Heidelberg, Germany). A glass/Teflon Potter Elvehjem tissue grinder or glass/glass grinder were used. Homogenates were divided into two portions: the one for the BDNF ELISA was supplemented with 2% BSA, 1M NaCl and 2% Triton X-100. The supplemented and non-supplemented portions were incubated on ice for 30–60 minutes and centrifuged at 11 600×g for 30 min at 4°C. Immediately afterwards BDNF ELISA was performed following the manufacturer’s instructions (Millipore, Billerica, MA, USA). The supernatants from the non-supplemented extracts were used for Western blotting analysis (BDNF, KCC2) and for ELISA (GABA) (Labor Diagnostika Nord, Nordhorn, Germany). All samples were run in duplicate.