Ab108249

Tissue and cell lysates were subjected to SDS-PAGE for separation of proteins followed by immuno-blotting as previously described [18 (link)]. Anti-ABAT (1:10,000 dilution; ab108249, Abcam, Cambridge, MA, USA) and anti-GAPDH (1:5000 dilution; 10494-1-AP, Proteintech, Chicago, IL, USA) were used to detect the protein levels of ABAT and GAPDH, respectively.

Liver homogenates and mitochondrial fractions were obtained in RIPA buffer (Sigma-Aldrich, SLM, USA) and subjected to denaturing SDS-PAGE under reducing conditions. Total protein concentrations were determined by the Bradford method and equal amounts (30 μg) were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and blocked for 1 h in TBST buffer (20 mM TRIS, pH 7.5; 500 mM NaCl; 0.5% Tween 20) containing 5% nonfat milk. Membranes were then washed and incubated in the presence of mouse anti-GABA-T antibody (Rb mAb to ABAT, ab108249, Abcam, Cambridge, UK) diluted 1/10,000 in TBST overnight at 4°C. As controls, in the case of homogenates, membranes were incubated in the presence of mouse anti-tubulin antibody (ab56676, Abcam, Cambridge, UK) diluted 1/1,000 or in the case of mitochondrial fractions in the presence of rabbit anti-VDAC1/Porin antibody (ab15895, Abcam, Cambridge, UK) diluted 1/1,000. After washing, membranes were incubated with secondary antibodies conjugated to alkaline phosphatase (1/5,000). Bands were revealed using AP conjugate substrate kit (Bio-Rad, CA, USA). Densitometric analysis was performed using the Image Lab Software (v 3.0, Bio-Rad, CA, USA).