Mouse anti glua2

For AMPAR Co-IP 2 mg total protein was incubated with 4 μg of either mouse anti-GluA1 (Clone RH95; Millipore), mouse anti-GluA2 (Clone 6C4; Millipore), or control mouse IgG1 (Clone G3A1; Cell Signaling Technology) antibodies overnight at 4°C, with gentle rocking. Immune complexes were precipitated for 2 h at 4°C using 20 μl protein A/G agarose beads (Santa Cruz Biotechnology). For NSG2-mC Co-IP, 1.5 mg total protein was incubated with either 50 μl anti-RFP mAb-agarose (MBL International Corporation) or RFP-Trap_M (ChromoTek Inc.) according to manufacturer’s instructions. Agarose beads from the above reactions were washed and denatured in 50 μl 1% SDS, and 20 μl was loaded on an SDS-PAGE gel for Western blotting or 2 μl of the supernatant was subject to capillary electrophoresis.

The following antibodies were used for western blotting: mouse anti-Synaptotagmin (1:250; Abcam, Cambridge, UK); rabbit anti-GluA1 (1:1000, Thermo Fisher Scientific, Dreieich, Germany); mouse anti-GluA2 (1:1000; Millipore, Schwalbach, Germany); mouse anti-NR1 (1:1000; BD Biosciences, San Jose, CA); rabbit anti-NR2A (1:1000; Abcam, Cambridge, UK); mouse anti-NR2B (1:1000; Abcam, Cambridge, UK); mouse anti-PSD-95 (1:1000; Thermo Fisher Scientific, Dreieich, Germany); mouse anti-β-actin (1:5000; Sigma, Taufkirchen, Germany); mouse anti-GAPDH (1:5000; GeneTex, Irvine, CA), peroxidase-conjugated goat anti-mouse and goat anti-rabbit (1:15,000; Dianova, Hamburg, Germany).