Figure 2.

Stimulation of hippocampal neurons with BDNF decreases the interaction of hnRNP K with a large number of transcripts. A, B, List of the 10 most significantly enriched biological processes associated with mRNAs bound to hnRNP K (A) and those that were regulated by BDNF (50 ng/ml for 10 min) (B), identified with the PANTHER classification system. Only categories showing at least a 2-fold enrichment (considering the size of our lists) were analyzed and the 10 categories displaying the highest –log₁₀ (p value) are shown. The number of transcripts belonging to each category and the fold change (designated in parenthesis) are indicated within graph bars. reg., regulation; pres., presynaptic; chem, chemical; syn., synaptic; pos., positive; proj., projection; diff., differentiation; neg., negative; sys., system; devel., development. C, Cultured hippocampal neurons were stimulated or not with BDNF (50 ng/ml) for 10 min before preparation of cellular extracts. hnRNP K was immunoprecipitated from control and BDNF-treated hippocampal neuron homogenates, and the associated transcripts were identified by microarray analysis. The specificity of transcripts associated with hnRNP K was assessed by subtracting the levels of correspondent mRNAs pulled down together with mouse IgG antibodies. hnRNP K-associated mRNAs were then compared between control and BDNF treated neurons. The results were obtained from the quantification of four different experiments performed in independent preparations, and are expressed as -log (p value) and log fold change (BDNF vs control). A total of 9509 transcripts showed a decrease in the interaction with hnRNP K in cells stimulated with BDNF; p < 0.05 (gray dots) as determined by the paired Student’s t-test. D, Stimulation of cultured hippocampal neurons with BDNF does not affect total hnRNP K protein levels. Hippocampal neurons were stimulated with BDNF for 10, 20, and 30 min, and the cellular extracts were analyzed by Western blotting. β-Actin was used as loading control. The results represent quantification of three independent experiments, and are expressed as percentage (mean ± SEM) of control. ns, nonsignificant as determined by ANOVA followed by Dunnett's multiple comparison test.