FIGURE 3.

TRIM2 binding to Bim following preconditioning ischemia is dependent on Bim phosphorylation by p42/p44 MAPK. A, schematic shows the rapid ischemic tolerance paradigm. Neuronal cultures were subjected to preconditioning ischemia (30-min OGD PC) 1 h prior to harmful ischemia (120 min OGD). B, viability was assessed in cultures subjected to 30-min, 120-min, or 30-min OGD followed by 1-h recovery and a subsequent 120-min OGD. Cell death was assessed by propidium iodide assay 24 h following the last ischemic event. Propidium iodide-positive cells are expressed as the percent of total (DAPI-stained) cells in the culture. Data shown are mean ± S.E. (error bars), n = 12. *, p < 0.001 versus control; **, p < 0.001 versus 120-min OGD (one-way ANOVA with the Bonferroni post hoc test). C, cells were subjected to 30-min OGD preconditioning ischemia and then recovered in the presence of 10 μm MG132 (MG) or 10 μm U0126 (U) for 1 h. TRIM2, Bim, and phospho-MAPK levels were assessed using immunoblotting. D, cells were subjected to 30-min OGD preconditioning ischemia and then recovered in the presence of 10 μm MG132 or 10 μm U0126 for 1 h. Lysates were incubated with GST-Bim and then subjected to TRIM2 immunoprecipitation (TRIM2 IP). Precipitated proteins were immunoblotted with a GST-specific antibody. Binding of Bim with TRIM2 increases following 30-min OGD and is reduced by U0126. E, cells were subjected to 30-min preconditioning ischemia. Lysates were prepared and incubated with GST-Bim or GST-3ABim. Following TRIM2 immunoprecipitation (TRIM2 IP), precipitated proteins were immunoblotted with a GST-specific antibody. The pulldown of GST-Bim from cell lysates by TRIM2 (TRIM2 IP) is increased following preconditioning ischemia. Nonphosphorylatable 3ABim did not pull down.