Hrp conjugated secondaries

Whole brains were extracted from PBS-perfused Gpr62 wild-type and knockout mice and dounce homogenized in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA and 0.5 mM EGTA) with complete protease inhibitors (Roche). Lysates were clarified at 21,500 x g at 4 °C for 10 min and the supernatants diluted with Laemmli Sample Buffer. Twenty microgram of protein per sample was run on NuPage Bis-Tris gels (ThermoFisher) and transferred to PVDF (Millipore). Blots were blocked in 5% skim milk powder in tris buffered saline with 0.1% Tween-20 and probed with mouse anti-beta-tubulin (Developmental Studies Hybridoma Bank E7), anti-MBP (Millipore MAB386) or anti-PLP (clone AA3 [32 (link)]) and appropriate HRP-conjugated secondaries (Cell Signaling). Blots were imaged on a G:Box gel imaging system (Syngene) and densitometry analysis performed using Image J software [31 (link)].