Rabbit anti albumin

Frozen cortices dissected from the half-brain of control rats and rats that experienced SE were homogenized on ice in 1.5 ml RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing a mixture of protease and phosphatase inhibitors (Roche Applied Science, Penzberg, Germany). The homogenates were placed on ice for 2 h and then centrifuged (14,000 ×g, 15 min, 4 °C). The protein concentration in the supernate was measured by Bradford assay (Thermo Fisher Scientific, Waltham, MA). The supernates (20 μg protein each) were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto PVDF membranes (Millipore). Membranes were blocked with 5% (w/v) non-fat milk at 25 °C for 4 h and then incubated overnight at 4 °C with the primary antibody: rabbit anti-albumin (1:2000, Abcam, Cambridge, UK) and mouse anti-β-Actin (1:1000, Abcam). This procedure was followed by incubation with Near-InfraRed dye-conjugated secondary antibodies (IR-680 and IR-800; 1:2000, Li-Cor, Lincoln, NE) at room temperature for 2 h. The blots were scanned using a ChemiDoc MP imaging system (BioRad, Hercules, CA). The band intensities were quantified by ImageLab 6.0.1 (BioRad).

Commercial antibodies included: rabbit anti-human transthyretin antibody (A0002, Agilent Dako, Santa Clara, CA), rabbit anti-BiP (Abcam, Cambridge, MA), rabbit anti-pIRE1a (Abcam, Cambridge, MA), rabbit anti-Ero-1 (Cell Signaling Technology, MA), rabbit anti-PDI (Abcam, Cambridge, MA), rabbit anit-LAMP 1 (Abcam, Cambridge, MA), rabbit anti-albumin (Abcam, Cambridge, MA) rabbit anti-ubiquitin (Santa Cruz, CA), rabbit anti-caspase-3 (Cell Signaling Technology, MA), rabbit anti-SQSMT1/p62 (MBL), mouse anti-β-actin (Cell Signaling Technology, MA), goat anti-rabbit or mouse HRP-conjugated IgG (Cell Signaling Technology, MA), donkey anti-rabbit Alexa 488 or 594 (ThermoFisher Scientific, MA). STF 083030 (an IRE1a inhibitor) was purchased from, Santa Cruz, CAS (307543-71-1)