Anti gad65

Cells were harvested, washed with PBS and cell lysis was performed in 50 mM Tris–HCl pH 7.4 buffer containing 1% Triton X-100, 150 mM NaCl, 0.5 mM EGTA, 0.5 mM EDTA and anti-protease cocktail (Complete Protease inhibitor Cocktail Tablets, Roche, France). Protein extracts (30 μg) were separated by SDS-PAGE and transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare, USA). The following antibodies were used for immunoblotting: anti-Nanog (Cell Signaling, 1:1000), anti-p21 (Santa Cruz Biotechnology, 1:200), anti-Actin (Millipore Chemicon, 1:10,000), anti-SSAR (Novus biological, 1:2000), anti-ABAT1 (GABA-T) (Sigma Aldrich, 1:2500), anti-GAD65 (Abcam, 1:500), anti-GAD67 (Abcam, 1:500), anti-HOT (Sigma, 1:2000), anti-GLUD1 (Sigma, 1:2000), and anti-SSADH (Novus Biological, 1:2000). The secondary antibodies were anti-mouse IgG (Santa Cruz Biotechnology, 1:10,000), anti-rabbit IgG (GE Healthcare, 1:10,000) and anti-goat IgG (Santa Cruz Biotechnology, 1:10,000). Signal detection was performed with the ECL + chemiluminescence detection system (PerkinElmer, France). Densitometric analysis was achieved using ImageJ software.

The neurons were fixed in 4% paraformaldehyde for 15 min, permeabilized for 10 min with 0.2% Triton X-100 in phosphate-buffered saline (PBS), and blocked with blocking buffer (5% bovine serum albumin and 0.2% Tween 20 in PBS). The neurons were incubated at 4 °C overnight with primary antibodies diluted in blocking buffer to the appropriate concentration. After washing with PBS, the neurons were incubated with secondary antibodies for 1 h at room temperature. Finally, the coverslips with neurons were mounted on slides with Fluoromount-G (SouthernBiotech, AL, USA). The following antibody dilutions were used: anti-PROX1 (1:500, Abcam, Cambridge, UK), anti-GAD65 (1:500, Abcam), anti-GFP (1:2000, Abcam), and all secondary antibodies (1:500, Invitrogen, NY, USA).