Figure 1.

Cul3 knockout strategy and validation in primary cultured hippocampal neurons. (A) Schematic representation of fCul3 mouse model and viral transduction Cre-LoxP knockout strategy. Exons 3–8 of Cul3 are represented along with loxP, neomycin-resistance, and frt (flippase recognition target) sites. Transduction of neurons with AAV-Cre-GFP results in recombination and loss of Cul3 exons 4–7. AAV-GFP transduction is used as a negative control (not depicted). (B) Experimental timeline of neuronal culture and AAV transduction. fCul3 mouse pups are sacrificed at P1, cultures transduced with AAV viruses at DIV10, then examined for outcome measures at DIV16, 6 days after transduction (ICC, immunocytochemistry). (C,D) Representative immunoblot [left, (C)] and graph of group data [right, (D)] for anti-CUL3,anti-β-actin and anti-RhoA blots following either AAV-Cre-GFP (WT-Cre, Het-Cre, Hom-Cre) or AAV-GFP (WT-GFP, Het-GFP, Hom-GFP) transduction in cultured fCul3 hippocampal neurons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ordinary two-way AVONA with Dunnett’s multiple comparisons test; bars represent mean ± SEM, 5 separately derived neuronal cultures.