Fig. 3.

Retrograde transport and nuclear localization of CREB2 requires NLS- importin interactions. (A) Hippocampal neurons (10–12 DIV, serum-containing medium) were incubated with NLS peptide (50 μg/mL) or mutant peptide (mNLS) for 1h, and stained with anti-CREB2 (green) and anti-MAP2 (red) antibodies. Nuclear CREB2 (green) was reduced with the NLS but not the mNLS peptide (P < 0.0001, Student's t test; n = 56 and 49, respectively, t = 4.1, df = 103). (B) Dendra-CREB2 was transfected into hippocampal neurons (9–10 DIV) and imaged after photoconversion of a small region in the dendrite (arrow). An increase in photoconverted Dendra-CREB2 (red) was observed in the nucleus within 3 min after photoconversion (arrowhead), with a reduction in the dendrite. Neurons transfected with Dendra-CREB2 were preincubated with NLS peptide or mutant peptide (mNLS) for 30 min before imaging. The percentage of photoconverted red signal that persisted at the site of photoconversion in the dendrite, and the percentage increase in the nucleus were quantified for all 3 conditions (line graph, n = 7, 11, and 9 for no peptide, NLS, and mNLS peptides respectively). The decrease in the dendrite and increase in the nucleus in the no peptide and mNLS peptide groups was statistically significant (P < 0.01) at all time points by ANOVA and Dunnett's posthoc multiple comparisons test (F = 7.19 and df = 24 for increase in nucleus; F = 119 and df = 30 for decrease in dendrite). [Scale bar, 10 μm (A); 20 μm (B).]