FIGURE 4.

Immunodepletion of TRIM2 from preconditioned neuronal lysates reduces Bim ubiquitination. Cell lysates from control and preconditioned cells (30-min OGD, 1-h recovery) were prepared and subjected to TRIM2 or control (CBP) immunodepletion. Antibodies raised against CREB binding protein or TRIM2 were bound to protein A/G-agarose beads and incubated with prepared lysates. A, to confirm removal of TRIM2 from the lysate, a sample (5%) was subjected to immunoblotting with a TRIM2-specific antibody. B, control and TRIM2-immunodepleted lysates were then used to measure Bim ubiquitination. Cell lysates were incubated with GST-Bim, biotinylated ubiquitin, and E1 and E2 proteins. GST-Bim was precipitated by GST pulldown, and the ubiquitinated precipitated proteins were immunoblotted with a biotin-specific antibody. Preconditioning results in Bim ubiquitination in control (CBP) immunodepleted lysates, but not TRIM2 depleted lysates. The white dotted line denotes where the image was cut and reassembled to match the above lane loading. C, quantification of three independent experiments is shown.