Clone e 4

In addition to the NRG2 antibodies described above, commercial and custom antibodies against the following proteins were used in this study: ErbB4, rabbit monoclonal mAB10 (1 μg ml⁻¹; ref. 7 (link)) and rabbit polyclonal custom antibody 5721 (unpublished; 0.4 μg ml⁻¹); Kv2.1, mouse monoclonal (clone K89/34; NeuroMab; 1 μg ml⁻¹); rabbit polyclonal antibody against NRG1 (SC-348; Santa Cruz Biotechnology; 0.2 μg ml⁻¹); anti-V5 epitope tag (AbD Serotech; 1 μg ml⁻¹); Bassoon, rabbit polyclonal (Synaptic Systems; 1:1,000); Gephyrin, mouse monoclonal (clone mAb7a; Synaptic Systems; 1:500); PSD95, mouse monoclonal (clone 7E3-1B8; Pierce; 1:500); Calbindin, mouse monoclonal (clone CB-955; Sigma; 1:1,000); GFP, rabbit polyclonal (Molecular Probes; 1:2,000); Erk2, rabbit polyclonal (C-14; Santa Cruz; 0.2 μg ml⁻¹); phospho-Erk2, mouse monoclonal (clone E-4; Santa Cruz; 0.2 μg ml⁻¹); GAPDH, mouse monoclonal (clone 6C5; Santa Cruz; 0.2 μg ml⁻¹); clathrin heavy chain, mouse monoclonal (SC-12,734; Santa Cruz; 0.1 μg ml⁻¹); GluN2A, rabbit monoclonal (clone A12W; Millipore; 1:2,000); GluN2B (clone 13; BD Biosciences; 1 μg ml⁻¹); and GluA1 (AB1504; Millipore; 0.2 μg ml⁻¹).

For the immunocytochemical experiments (IF), neurons cultured on coverslips and stimulated for 10 min with 2 mM CaCl₂ or 2 µM TG in 0.5 mM EGTA were fixed in ice-cold 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS) for 10 min at room temperature. After permeabilization in 0.1% Triton X-100 and blockade with 2% normal donkey serum (NDS) in PBS for 30 min, an antibodies against rabbit STIM1 (1:50, ProteinTech Group) or mouse STIM1 (1:25, clone CDN3H4, Abnova), rabbit STIM2 (1:50, Alomone Labs), mouse NR2A (1:25, clone E-4, Santa Cruz Biotechnology), mouse NR2B (1:50, clone N59/36, Abcam, Cambridge, UK) and chicken MAP2 (1:500, Invitrogen) diluted in 2% NDS were applied for 2 h at room temperature. The staining was detected using anti-mouse Alexa Fluor 488-, anti-chicken Alexa Fluor 568- and anti-rabbit Alexa Fluor 647-conjugated secondary antibody (Invitrogen) in blocking solution for 45 min at room temperature. To visualize the nuclei of cells, we included the Hoechst 33342 dye (Invitrogen) in the wash. Coverslips were mounted on slides with ProLong Gold Antifade Mountant (Invitrogen).