Ab1551

Hippocampi were homogenized by sonication in 50 mM Tris pH 7.4/2% SDS followed by incubation at 95 °C for 5 min. Extracts were run in duplicate and resolved by denaturing PAGE and blotted to nylon membrane (Immobilon-FL, Millipore). Blocking and antibody incubations were for 1 h at room temperature in Tris-buffered saline, 0.05% tween 20, 3% non-fat dry milk. Washes were carried out TBS-Tween without milk. Proteins were detected using commercially available antibodies against mGluR1 (Millipore AB1551), mGluR5 (Millipore AB5675), and α-tubulin (Sigma T9026) in conjunction with dye conjugated anti-mouse or rabbit secondary antibodies (Li-COR, Lincoln, NE). Blots were scanned and integrated band intensities determined using an Odyssey infrared imaging system. mGluR1 or mGluR5 band intensities were normalized to the band intensity for tubulin in that lane as a loading control and data were presented as percent of the average normalized control value.

Proteins were separated by SDS-PAGE and transferred to PVDF membrane for Western blotting. Membranes were blocked in 5% w/w non-fat milk powder in PBS-T. Primary antibodies used for blotting were: MEF2A (rabbit monoclonal, 1:5000, Abcam ab109420), GluA1 (rabbit polyclonal, 1:1000, Millipore AB1504), GluA2 (rabbit polyclonal, 1:2000, Synaptic Systems 182103), GAPDH (mouse monoclonal [6C5], 1:10000, Abcam ab8245), Arc (rabbit polyclonal, 1:1000, Synaptic Systems 156003), mGluR1α (rabbit polyclonal, 1:500, Millipore AB1551), mGluR5 (rabbit polyclonal, 1:5000, Upstate Biotechnology 06–451). For protein detection, membranes were incubated with HRP-conjugated secondary antibodies and visualised by enhanced chemiluminescence. Protein bands were quantified by densitometry using ImageJ (NIH). For total protein expression, band intensity of the protein of interest was normalised to the loading control. For proportional surface expression, surface band intensity of the protein of interest was normalised to its band intensity in the total lysate.