Figure 7.

NGF promotes the migration of p65/RelA to the nucleus, a process necessary for the increase of Hes1 and Hes5 mRNA. (A) Hippocampal neurons were plated at an initial density of 30,000/cm². After 7 DIV they were for 3.5 h incubated in NGF, SN50 (an inhibitor of NF-kappaB migration into the nucleus, used here at 28 μg/ml), p75-antibody, NGF plus SN50, or NGF plus p75-antibody (control, no treatment) and then processed to obtain nuclear extracts. The same amount of nuclear protein was separated by SDS/PAGE and transferred to nitrocellulose membranes, which were subsequently probed with an anti-p65 antibody. Note the presence of p65 in the nucleus after treatment with NGF (arrowhead). Both SN50 and p75-antibody prevented the NGF-induced appearance of p65 in the nucleus. Long arrows point to nonspecific bands, asterisks to the heavy chain of the p75-antibody. (B) The intensity of the p65 band was determined by densitometry. Shown is the quantitative analysis from three different experiments. (C) Cultured hippocampal neurons were treated with NGF, with NGF plus SN50, or with SN50 alone. The presence of the inhibitor of the nuclear translocation of NF-kappaB prevented the NGF-induced increase of both Hes1 and Hes5 mRNA.