Detailed information is provided in Supplement 1. Briefly, solubilized protein (10–60 μg per lane) was subjected to electrophoresis, transferred to nitrocellulose membranes and stained with Memcode Reversible Protein Stain Kit (Thermo Fisher Scientific). The membranes were blocked in blocking buffer and incubated at 4°C overnight with primary antibodies. Rabbit polyclonal antibodies were used against Homer 1, PSD-95 [1:5000;1:2000; Synaptic Systems GmbH (SYSY) Goettingen, Germany], or mGluR5 and GluA1 (1:200;1:1000 Millipore (Upstate), Billerica, MA) or dynamin-3 (1:1000; Abcam). In addition mouse monoclonal antibodies were used against GluN1 (114 011, 1:1000, SYSY) and GAPDH (MAB374, 1:60,000 Millipore (Upstate)). Membranes were incubated with goat anti-rabbit or goat anti-mouse IRDye 680 or IRDye 800 secondary antibodies (LI-COR, Lincoln, NE, USA). Each protein was analyzed as a single or a double band based on predicted molecular size 45 (Homer 1), 95 (PSD95), 130 (mGluR5), 106 (GluA1), 110 (GluN1-analysed as a double band), 100 (dynamin-3) and 35 (GAPDH) kDa. GAPDH and/or Memcode optical density were used to control for total protein content. Membranes were developed with the LI-COR infrared imaging system (LI-COR) and images quantified using average integrated intensity values.
