The Journal of Neuroscience, May 9, 2007, ():
EphA4 Signaling Regulates Phospholipase C
1 Activation, Cofilin Membrane Association, and Dendritic Spine Morphology
J. Neurosci. Zhou et al.
27: 5127
Supplemental Data
Files in this Data Supplement:
- supplemental material -
Supplemental Materials and Methods
- supplemental material
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Supplemental Figure 1. Knock-down of PLC?1 expression using RNAi. A pSUPER plasmid inducing the expression of a short-hairpin RNA directed against PLC?1 and driving EGFPf was used for RNAi in NIH3T3 cells. The sequence used for RNAi has been previously characterized and shown to selectively knock-down PLC?1 expression, while leaving the expression of the closely related family member, PLC?2, intact (Patterson et al., 2002). A plasmid that drives the expression a scrambled sequence of the PLC?1 shRNA and EGFPf was used as a control. To verify the efficacy of the PLC?1 RNAi, 10?g of control or PLC?1 shRNA plasmid was transfected into NIH3T3 cells. 72 hours following transfection, the cells were lysed and the level of PLC?1 protein detected by western blot. Blotting for GAPDH verified approximate equal loading between lanes. In the mock transfection lane, cells were subjected to the same experimental conditions except the DNA was omitted.
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Supplemental Figure 2. EphA4 and PLC?1 partially co-localize in CA1 pyramidal cell dendritic spines. Max projection of an EGFPf-infected organotypic hippocampal CA1 pyramidal neuron apical dendrite (green) immunolabeled for EphA4 (red) and PLC?1 (blue). (TOP) Shown are individual channels in grayscale for EphA4, PLC?1 and EGFPf, respectively. (BOTTOM) Merged image showing a composite of the three channels above. Masked image refers to the same field shown to the left following a three-dimensional "masking" procedure to preserve only the EphA4 and PLC?1 punctae found within the volume of the dendrite. High magnification images show spines that reveal co-expression of both EphA4 and PLC?1. Asterisks label example spines that have EphA4 only (or low levels of PLC?1) and arrowheads show spines that do not have (or have low levels of) EphA4 or PLC?1. Scale bars, 1 µm.
