Fig. 3: Bi-directional redistribution of DISC1/PSD-95/kalirin-7 signalosome by 17β-estradiol (E2).

a Whole cell lysate, crude synaptosome (P2) and cytosol (S2) fractions of mixed sex cortical neurons treated with 17β-estradiol (E2) for 0 or 30 min analyzed by western blotting for expression of DISC1, PSD-95 or kalirin-7; β-actin was used as a loading control. Arrows indicate DISC1 and kalirin isoforms measured in analysis. E2 has no effect on overall expression levels of DISC1, kalirin-7 or PSD-95 within 30 min as seen in whole cell lysate. Treatment with E2 causes a reduction of DISC1 in crude synaptosome fraction and an increase in cytosolic fraction. E2 treatment resulted in an enrichment of kalirin-7 and PSD-95 within crude synaptosome fraction and concurrent reduction in cytosolic fraction. b Quantification of DISC1, PSD-95 and kalirin-7 enrichment in crude synaptosome and cytosol fractions. c GFP-expressing and DISC1 (440) stained cortical neurons treated with E2 for 30 min or not. Quantification of DISC1 cluster intensity within spines reveals a reduction in intensity within spines after E2 treatment. d Cortical neurons (DIV 25) fixed before and after treatment with E2 and immunostaining for kalirin-7 and PSD-95. E2 treatment (30 min) increases kalirin-7 puncta density (black bars). Assessment of number of kalirin-7/PSD-95 co-localized puncta revealed and increase in kalirin-7 and PSD-95 positive puncta (red bars), indicating that kalirin-7 is being targeted to synapses (*p < 0.01 (corrected for multiple comparisons) Student t-test; n = 12–15 cells per condition from 3 independent cultures). e GFP-expressing and kalirin-7 stained cortical neurons treated with E2 for 30 min or not. Quantification of kalirin-7 cluster intensity within spines reveals an enrichment within spines following treatment (*p < 0.05, Student t-test; n = 270–341 spines from 6 cells per condition from 3 independent cultures). Scale bar = 5 µm.