Ab108404

Frontal cortex, anterior pituitary, and posterior pituitary from 3-mo-old Sprague Dawley rats were homogenized separately with a Dounce homogenizer in 300 µl of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% IGEPAL CA-630; I8896; Sigma-Aldrich), with protease inhibitor cocktail (P8340; Sigma-Aldrich) and 1 mM PMSF. The homogenates were sonicated for 5 min on ice and centrifuged at 16,000 g for 20 min in 4°C to collect the supernatant. Protein concentrations were determined by BCA assay and adjusted to 1 µg/µl for all samples. A 15-µl aliquot of each sample was resolved on 15% SDS-PAGE gel, transferred to nitrocellulose membranes, and probed with rabbit anti-calbindin D28K (1:1,000, ab108404; Abcam), rabbit anti-calretinin (1:500, ab92341; Abcam), and rabbit anti-parvalbumin (1:1,000, ab11427; Abcam) antibodies, followed by secondary horse radish peroxidase conjugated antibody, and developed with enhanced chemiluminescence (Pierce Chemicals). The same membrane was then stripped with stripping buffer (1.5% glycine, 0.1% SDS, and 1% Tween 20, pH 2.2) for 15 min at room temperature, probed with mouse anti-actin (1:2,000; Abcam), goat anti-oxytocin (1:1,000, EB09854; Everest), or guinea pig anti–growth hormone antibodies (1:2,000). The experiments were repeated twice.