Figure 4.

Knockdown of CaMKK and CaMKIγ protein expression inhibits axonogenesis and decreases axonal length. A, E18 hippocampal neurons were electroporated before plating with pMU6pro alone (control) or pMU6pro containing shCaMKK, shCaMKIα, shCaMKIβ, shCaMKIδ, or shCaMKIγ. Between 2 and 48 h in culture, low-density cultures of electroporated neurons were incubated with 20 μm SP600125 to suppress intrinsic neuronal polarization to achieve effective knockdown with the plasmid based shRNA (see Results). At 48–72 h in culture, electroporated neurons were released from SP600125 block by transfer of coverslips to new glial culture plates for an additional 48 h. Representative images of neurons fixed and stained with Tau-1 at 96 h are shown. The third from top panel shows staining from CaMKIα shRNA. This image is also representative of neurons transfected with shCaMKIβ or shCaMKIδ. B, Summary of three to five independent experiments. C, Average axonal length as measured by length of primary process containing Tau-1 staining from three independent experiments. Scale bars: 20 μm (all panels).