FIGURE 2:

Knockdown of Dock4 suppresses dendritic spine formation. (A) Cell lysates from HEK293T cells cotransfected with indicated shRNAs (ineffective shControl or effective shDock4 #1, #2) and Flag-tagged, wild-type Dock4 (Dock4-WT) or shDock4 #1-resistant form of Dock4 (Dock4-res) were analyzed by immunoblotting with antibodies against Flag and α-tubulin. (B) Knockdown of endogenous Dock4 in hippocampal neurons. Primary cultured rat hippocampal neurons were cotransfected with EGFP and the indicated shRNA expression vector at 11 DIV. At 4 d after transfection, neurons were fixed and stained with antibody against Dock4 (magenta). Arrowheads indicate the transfected cells. Scale bar, 20 μm. (C) Quantification of the knockdown efficiency. Under the same conditions as those in B, the Dock4 fluorescence intensity in the cell bodies of the EGFP-positive transfected cells relative to that of nearby untransfected cells was measured. The data are mean ± SE of 10 cells. (D–G) Cultured hippocampal neurons were cotransfected with EYFP and the indicated shRNA expression vector at 11 DIV and then fixed at 15 DIV. Morphology of dendrites is shown by EYFP fluorescence. Scale bar, 5 μm. (H) Under the same conditions as those in D–G, the dendritic protrusions per 10 μm of dendrite were counted. The data are mean ± SE of 45 cells in three independent experiments. *p < 0.05; ***p < 0.001 (vs. shControl).