Fig. 4
CARMIL3 localization during synapse development. a Schematic depicting HITI labeling of endogenous CARMIL3 with myc or smFP. b Representative western blot of myc pulldown of endogenous CARMIL3 at P7, P14, P21, and P28. c Expression of endogenous CARMIL3 in the P14 mouse brain. CARMIL3 expression is most pronounced in the cortex (green—Cas9-2A-GFP, blue—DAPI). Scale bar is 900 μm. d Representative images of HITI-mediated mCherry (magenta) expression in the CA1 hippocampus. Scale bars are 25 μm. e Representative images of HITI-mediated mCherry (magenta) expression in the cortex. Scale bars are 25 μm. f Control image of AAV-HITI-smFP-P2A-mCherry expression in cultured hippocampal neurons without CARMIL3 sgRNA. Neurons were co-transfected with a GFP fluorescent fill (green) and infected with AAV-Cas9. Scale bar is 5 μm. g Knock-in expression of CARMIL3-smFP (gray) in cultured neurons using CARMIL3 sgRNA. CARMIL3 localizes along dendrites and dendritic protrusions in hippocampal neurons and neurons are marked with mCherry expression (blue) under the control of the endogenous CARMIL3 promoter. Neurons were also co-transfected with a GFP fill (green) and infected with AAV-Cas9. Scale bar is 5 μm. h IMARIS reconstruction of a dendritic section of a control hippocampal neuron. Dendrite is gray, protrusions are blue, and smFP is green. Scale bar is 5 μm. i IMARIS reconstruction of dendritic section of a CARMIL3-smFP KI neuron. Dendrite is gray, protrusions are blue, and CARMIL3-smFP is green. Scale bar is 5 μm
