Western Blot Analysis of GGT in Immune Cells and Exosomes

For western blot determinations of GGT, isolated monocytes, lymphocytes, platelets and cultured endothelial HMEC-1 cells—harvested in hypotonic lysis buffer (10 mM Tris–HCl, pH 7.8)—or plaque derived exosomes were used. Non-adherent lymphocytes were isolated from plated PBMCs, whereas platelets were obtained from buffy-coat derived platelet concentrates. All samples were thoroughly washed in order to remove contaminating plasma GGT. Endothelial HMEC-1 cells were grown in MCDB 131 medium (Life Technologies) supplemented with 1 µg/ml hydrocortisone, 10 ng/ml EGF (Life Technologies) and 10 % v/v fetal calf serum, and cultured at 37 °C in a 5 %/95 % CO₂/air atmosphere.
All samples were separated by 8 % SDS-PAGE [37 (link)] and incubated with rabbit anti-GGT IgG directed against the C-terminal 20 amino acids of human GGT heavy chain prepared as described [36 (link)]. Visualization of protein bands was obtained using a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ECL detection system (Roche, Basel, Switzerland). Bands were analyzed with a Bio-Rad ChemiDoc apparatus equipped with the QuantityOne software.