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. 2008 Nov 26;28(48):12725–12735. doi: 10.1523/JNEUROSCI.4619-08.2008

Figure 2.

Figure 2.

The 115 kDa fragment of NR2B produced by calpain cleavage is increased in YAC128 tissue, specifically in the striatum. A, Top panel, Western blots showing lysates of striata and cerebella from 2 month-old YAC and WT mice probed with an antibody raised against NR2C (top strip), which recognizes all NR2 subunits. Indicated are the bands representing full-length NR2B/2A, NR2C, and the 115 kDa calpain-cleaved fragment of NR2B. Blots are reprobed with antibodies against NR1 (middle strip) and actin (bottom strip) and are representative of 3 independent experiments. Bottom panel, Control showing that when cultured YAC72 MSNs are treated with 1 μm calpain inhibitor-1 (CI-1) for 24 h before harvesting, the 115 kDa band is markedly reduced, as detected with the anti-NR2C antibody. Blot is representative of n = 7 independent experiments with 4 different culture batches of YAC72 and 3 different batches of WT MSNs. B, NR2 subunits were immunoprecipitated (IP lanes) from 2-month-old WT and YAC128 striatal tissue using the anti-NR2C antibody, and Western blots were probed with an antibody against the N-terminal region of NR2B. Note the bands representing full-length and the 115 kDa fragment of NR2B in both IP and lysate (Lys) lanes, and that the ratio of NR2B fragment to full-length protein is higher in YAC128 than WT striatal tissue. Blot is representative of 4 paired experiments from 4 different mice of each genotype. C, Graph showing the band densities averaged from 3 experiments as shown in A (striatum and cerebellum were dissected from 1–2 mice for each genotype in each independent experiment), expressed as a ratio of 115 kDa NR2B fragment to full-length NR2B. This ratio was significantly higher in YAC128 than in WT striatum (*p < 0.05 by unpaired two-tailed Student's t test). There was no difference between genotypes in the cerebellum. D, Data from the same blots, expressed as a ratio of 115 kDa NR2B fragment to NR1. Again, this ratio was significantly higher in YAC128 striatum than WT striatum (**p < 0.01 by unpaired two-tailed Student's t test), and there was no difference between genotypes in the cerebellum.