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. Author manuscript; available in PMC: 2014 Feb 25.
Published in final edited form as: Neurobiol Dis. 2012 Aug 25;0:169–176. doi: 10.1016/j.nbd.2012.08.013

Figure 6. Interaction of Tat immune complexes with the NMDA receptor, but not kainate receptors.

Figure 6

(A) Lanes 1–6: Protein G sepharose beads were prepared with a monoclonal anti-NR1 antibody. HEK 293 cells expressing NR1A and NR2A were incubated for 1 hour in conditioned media with the indicated agent(s): untreated (lane 1), Tat protein (lane 2), nitrosylated Tat (lane 3), TatΔ31–61 (lane 4), Tat and anti-Tat antibody (lane 5), Tat and anti-Tat antibody and NMDA (lane 6). The cells were then harvested for immunoprecipitation over the anti-NR1 loaded protein G sepharose. Eluted proteins were separated by SDS-PAGE and immunoblotted with anti-Tat antibody. As controls, NR1 (lane 7) and Tat (lane 8) were run on the gel. (B) Top panel: Tat protein or vehicle was incubated with HEK 293 cells which had been transfected with NMDA receptor proteins, NR1A and NR2A. Immunoprecipitation was performed with anti-Tat antibody. Eluted proteins were immunoblotted with anti-NR1A antibody. Bottom panel: Tat protein or vehicle was incubated with HEK 293 cells which had been transfected with an AMPA receptor-GFP fusion protein, GluR1-GFP. Immunoprecipitation was performed with anti-Tat antibody. Eluted proteins were immunoblotted with an antibody against the GluR1-GFP. As a control, the third lane, shows cell lysate from HEK 293 cells transfected with NR1A and NR2A (top panel) and with GluR1-GFP (bottom panel). The arrows indicate the monomeric forms of NR1A (top) and GluR1 (bottom). (C) Mixed rat neuronal cultures were exposed to NMDA and/or anti-NR2b antibody. Mitochondrial membrane potential was measured 18 hours later. NMDA alone caused toxicity versus untreated controls (p<0.001). When the cells were pre-treated with anti-NR2b antibody for 30 minutes prior to addition of NMDA, it provided significant protection versus the toxicity seen with NMDA alone (*p<0.05, **p<0.01). Concentrations: NMDA 125 μM, ab1 5ng/μl, ab2 10ng/μl. Data represents mean ± SEM of at least three independent experiments, analyzed by ANOVA with Neumann-Keuls post-test.