Gat 3

The animals were sacrificed by decapitation and the cerebellums were dissected and homogenized in 66 mM Tris–HCl (pH 7.4), 1 % SDS, 1 mM EGTA, 10 % glycerol, 1 mM sodium ortho-vanadate, and 1 mM sodium fluoride containing protease inhibitor cocktail (Roche, Mannheim, Germany). Samples were subjected to electrophoresis and immunoblotting as in Felipo et al. [42 (link)]. Primary antibodies were against IL-4, IL-10, iba-1, Ym-1, and GAT 3 (1:2000) from Abcam (Cambridge, UK); IL-1β (1:500) from R&D SYSTEMS (Minneapolis, USA); Arg-1 from BD Bioscience (NJ, USA); and glial fibrillary acidic protein (GFAP) (1:5000) from Sigma (St. Louis, MO, USA). As a control for protein loading, the same membranes were also incubated with anti-actin (Abcam, Cambridge, MA; 1:1000). Secondary antibodies were anti-rabbit, anti-goat, or anti-mouse IgG (1:2000) conjugated with alkaline phosphatase (Sigma, St. Louis, MO). The images were captured using the ScanJet 5300C (Hewlett- Packard, Amsterdam, the Netherlands) and band intensities quantified using the Alpha Imager 2200, version 3.1.2 (AlphaInnotech Corporation, San Francisco).

After five weeks of golexanolone treatment, cerebellum and hippocampus were dissected from six rats per group and homogenized in 50 mM TRIS–HCl pH 7.5, 50 mM NaCl, 10 mM EGTA, 5 mM EDTA and protease and phosphatase inhibitors. Thirty μg of protein was loaded in a 15% SDS gel and immunoblot was performed with antibodies against: GFAP (Sigma, 1:5000), IBA1 (Abcam, 1:500), CCL2 (Proteintech, 1:200), TNF‐a (R&D SYSTEMS, 1:500), Glutaminase (Novus, 1:1000), GAT‐3 (Abcam, 1:1000), TrkB (Abcam, 1:500), GAD67 (Abcam, 1:500) and GABA_A beta3 subunit (Abcam, 1:1000). β‐Actin (Abcam, 1:5000) or GAPDH (Millipore, 1:15,000) were used as a control for protein loading. For plasma proteins analyzed by Western blot we used Coomassie R‐350 staining on the membrane, after immunoblotting,³⁵ as loading control. We selected a Coomassie‐stained band that was not saturated as representative of the whole protein lane. For quantification, intensity of plasma proteins was divided by intensity of complete protein lane, as is performed in Ref. [35 (link)].

This was analyzed as in Ref. [36 ]. Transversal 400 μm thick cerebellar slices were added to tubes containing ice‐cold Krebs buffer with or without 2 mM BS3 (Pierce, Rockford, IL) and incubated for 30 min at 4°C. Cross‐linking was terminated by adding 100 mM glycine (10 min, 4°C). The slices were homogenized in lysis buffer (66 mM Tris–HCl pH 7.4. 1% SDS, 1 mM EGTA, 10% glycerol, 0.2 mg/ml leupeptin, 1 mM NaF, 1 mM Na‐orto‐vanadate) by sonication for 20 s. Samples treated with or without BS3 were analyzed by Western blot using antibodies against TNFR1 (Abcam, 1:1000), P2X4 (Invitrogen, 1:500), TrkB (Abcam, 1:500), KCC2 (Millipore, 1:1000) and GAT‐3 (Abcam, 1:1000). The surface expression of these proteins was calculated as the difference between the intensity of the bands without BS3 (total protein) and with BS3 (non‐membrane protein).³⁷