Quantity analysis software

Total cell lysates from kidney were collected in PBS and resuspended in a modified radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), supplemented with protease inhibitor (Roche Diagnostics, Mannheim, Germany) and incubated at 4°C for 30 min. Cell lysates were centrifuged at 14,000 rpm in a microcentrifuge for 10 min at 4°C. The supernatants were collected, and the protein concentration was measured by Bio-Rad protein assay kit. 60 μg of total proteins was processed for western blotting. Rabbit polyclonal antibodies specific for LY6E (Santa Cruz) and rabbit polyclonal antibodies for β-actin (Sangon Biotech) were used. Quantity analysis software (Bio-Rad Laboratories) was used for density analysis. Quantitation of LY6E protein expression was evaluated by a FluorChem FC2 system (NatureGene Corp., New Jersey, USA). Data was expressed as the ratio of LY6E integral optical density and β-actin integral optical density in the same sample.

Equal amounts of protein were loaded on 10% sodium dodecyl sulfate polyacrylamide gels for electrophoresis. Separated proteins were then transferred on nitrocellulose membrane. GABA_Aα1, GABA_Aα2, GABA_Aα3, GABA_Aα5, GABA_Aβ2, Gp78, SYVN1, C/EBP homologous protein (CHOP) or GRP78 was detected with primary antibody at a 1:1000 dilution in TBS containing 5% non-fat dried milk and 0.05% Tween-20. After incubation with secondary antibody (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG) at a 1:2000 dilution, immunoreactive bands were detected with chemiluminescent substrate (RPN2232, GE Healthcare). The immunoreactive signals were quantified by quantity analysis software (Bio-Rad).