Figure 3.

CREB is sumoylated by PIAS1 in the hippocampus. A, Co-IP showing that PIAS1 is associated with CREB and vice versa in rat hippocampus. Identification of CREB sumo acceptors in rat hippocampus by Western blot against CREB (B) and SUMO1 (C). The quantified results are shown on the right. D, Confocal images showing EGFP-CREB transfection and expression in the CA1 area at different magnifications. Cells that show both green fluorescence (EGFP) and blue fluorescence (DAPI) are cells successfully transfected with the plasmid. Red arrows indicate the area of transfection. Scale bars: top, 500 μm; bottom, 100 μm. E, NMDA was injected into rat CA1 area bilaterally (1.2 μg/μl, 0.7 μl each side) and in vitro SUMOylation for CREB was performed 30 min later. In one group, the SUMO1 mutant protein was added to the reaction and, in another group, the ligase-deficient mutant Flag-PIAS1W372A plasmid was transfected. Effect of NMDA injection was confirmed by elevated level of pCREB from Western blot in a separate analysis. Flag-PIAS1W372A transfection was confirmed by Western blot against the Flag tag. n = 5 each group. The quantified result is shown on the right. F, Subcellular distribution of PIAS1, CREB, and the sumoylated form of CREB in the cytosol and nucleus fractions from the rat CA1 tissue. Data are expressed as mean ± SEM. **p < 0.01; #p < 0.001. Experiments were performed in duplicate or triplicate.