Calpain activity in NR1-immunoprecipitates from SKNBE cells.

<p>(A) SKNBE cells (10⁵) untreated (Control) or treated with 100 μM NMDA and 10 μM glycine for 2 hours (NMDA) were lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material was suspended in 100 μL of HEPES buffer and, after addition of 5 or 1000 μM CaCl₂, incubated at 37°C for 10 min in the presence of 50 μM t-Boc-Leu-Met-CMAC fluorogenic calpain substrate. Calpain activity was measured as described in Methods and the values are reported as difference between the fluorescence monitored at the indicated calcium concentration minus the one monitored in 1 mM EDTA. The values reported are the arithmetical mean ± SEM of four different experiments. (B) Calpain activity was assayed in NR1-immunoprecipitates from 10⁵ SKNBE cells treated as in (A) (Membranes). Intracellular calpain activity was also measured in 10⁵ SKNBE cells treated with 100 μM NMDA and 10 μM glycine for 2 hours (Intact cells).