1. Molecular Determinants for Subcellular Localization of PSD-95 with an Interacting K+ Channel 
Donald B Arnold, David E Clapham 
Neuron 
Volume 23, Issue 1, Pages (May 1999)
DOI: /S (00)

2. Figure 1
PSD-95:GFP Localizes to Dendritic Spines in Transfected Cortical Pyramidal Neurons
Two hundred micrometer thick slices cut from P12-P14 rats were transfected with alkaline phosphatase and PSD-95:GFP using biolistics and cultured for 40 hr prior to fixation and staining for alkaline phosphatase with a fluorescent antibody. (A) Alkaline phosphatase filled the entire cell homogeneously. The axon (marked with arrows) projects in a direction 180 degrees from the apical dendrite and extends far beyond the basal dendrites, allowing easy identification. Scale bar, 20 μm. (B) PSD-95:GFP (green; overlap with red, yellow) produces a punctate staining pattern in the dendrites that is distinct from the expression pattern of alkaline phosphatase. Scale bar, 10 μm. (C and D) Higher power image of area marked in yellow in (A). Scale bar, 5 μm. Alkaline phosphatase is present in the axon (arrows) while PSD-95:GFP (D) is absent. (E–G) Image of area outlined in green in (A). Scale bar, 5 μm. (E) In dendrites, PSD-95:GFP (green; overlap with red, yellow) is localized to puncta at the tips of spines. (F) Alkaline phosphatase alone. (G) PSD-95:GFP alone.
Neuron  , DOI: ( /S (00) )

3. Figure 2 PSD-95:GFP Colocalizes with an Excitatory Postsynaptic Marker
Cortical slices transfected with PSD-95:GFP were frozen, cut into sections with a thickness of 15 μm, and stained for α-actinin 2 and GFP. Each punctum of PSD-95:GFP overlaps with a punctum of α-actinin 2. Since α-actinin 2 is localized to excitatory postsynaptic sites, it is likely that PSD-95:GFP is as well. Note that there are more puncta of α-actinin 2 visible than of PSD-95:GFP because PSD-95:GFP is only in transfected cells, whereas α-actinin 2 is present in untransfected cells as well. Scale bar, 5 μm.
Neuron  , DOI: ( /S (00) )

4. Figure 3
Intensity of Fluorescence Related to PSD-95:GFP and to Alkaline Phosphatase as a Function of Distance from the Cell Body
(A) The intensity of PSD-95:GFP puncta did not vary systematically with distance from the soma. The line shown was calculated by linear regression. (B) Intensity of alkaline phosphatase staining diminished with increasing distance from cell body.
Neuron  , DOI: ( /S (00) )

5. Figure 4
At Least One PDZ Domain Is Necessary for Localization of PSD-95:GFP
(A) PSD-95 contains five modular domains. GFP was added to the C terminus. (B) PSD-95:GFP was able to localize to dendritic spines with PDZ1 deleted. Scale bar, 10 μm. (C–G) Deletion of up to two PDZ domains did not affect localization. The chart on the right indicates which PDZ domains have been deleted (-) or spared (+) for each construct tested. Scale bar, 5 μm. (H–K) ΔPDZ1.2.3PSD-95:GFP (all three PDZ domains deleted) did not localize. (H) Alkaline phosphatase and ΔPDZ1.2.3PSD-95:GFP. Scale bar, 10 μm. (I) ΔPDZ1.2.3PSD-95:GFP. (J) Alkaline phosphatase. Scale bar, 5 μm. (K) ΔPDZ1.2.3PSD-95:GFP.
Neuron  , DOI: ( /S (00) )

6. Figure 6
Ratio of Intensity of Puncta in Dendritic Spines to Intensity in the Adjacent Dendrite for PSD-95:GFP Mutants
PSD-95:GFP fluorescence was approximately 10 times more intense in puncta found in dendritic spines than at points on adjacent dendritic shafts for constructs where up to two PDZ domains were deleted and when the guanylate kinase (GK) domain alone was deleted. When all three PDZ domains were deleted and when the GK and SH3 domains were deleted, the ratio was not significantly different from 1. A ratio of 1 was calculated for alkaline phosphatase at identical points to PSD-95:GFP on the same cells. Each ratio was averaged over five cells and each ratio was from over 50 measurements. The error bar reflects the variability between cells.
Neuron  , DOI: ( /S (00) )

7. Figure 5
The SH3 Domain (Including Linkers) Is Necessary for Localization of PSD-95:GFP
(A–C) ΔSH3.GKPSD-95:GFP (SH3, GK domains and linkers between PDZ3 and SH3 [L1] and between SH3 and GK [L2] deleted) did not localize to dendritic spines. (A) Alkaline phosphatase and ΔSH3.GKPSD-95:GFP. Scale bar, 10 μm. (B) Alkaline phosphatase. Scale bar, 5 μm. (C) ΔSH3.GKPSD-95:GFP. (D–F) ΔGKPSD-95:GFP (GK domain deleted) localized to dendritic spines. (D) Alkaline phosphatase and ΔGKPSD-95:GFP. Scale bar, 10 μm. (E) Alkaline phosphatase. Scale bar, 5 μm. (F) ΔGKPSD-95:GFP.
Neuron  , DOI: ( /S (00) )

8. Figure 7 Kv1.4 Localizes PSD-95:GFP to the Axon
Kv1.4 tagged with a FLAG epitope was cotransfected with PSD-95:GFP. (A) Kv1.4 (red) is localized only to the axon. (B) PSD-95:GFP (green) is localized to the axon as well as the dendrites. (C) PSD:95-GFP and Kv1.4 are colocalized on the axon (yellow), but PSD-95:GFP alone is in the dendrite (green). (D) Camera lucida drawing of axon (yellow) and dendrite (green) of cortical pyramidal cell shown in A–C. Scale bars in A–D, 10 μm. (E) Higher power of highlighted axonal area in (D). Kv1.4 and PSD-95:GFP are colocalized in the axon. (F) Higher power of highlighted dendritic area in (D). PSD-95:GFP is localized to dendritic spines, but Kv1.4 is absent. Note that this image does not include the plane of the axon, and thus only the dendrite is visible. Scale bars in (E) and (F), 5 μm.
Neuron  , DOI: ( /S (00) )

9. Figure 8
Interaction with PSD-95 Is Necessary for Kv1.4 to Localize to the Axon
(A) Kv1.4 V655A (red, C-terminal valine changed to alanine) is localized throughout the cell, but not in the axon (arrows) when cotransfected with PSD-95:GFP. (B) PSD-95:GFP (green) is localized to dendritic spines but not to the axon when cotransfected with KV1.4 V655A. (C) Kv1.4 (red) is localized throughout the cell when cotransfected with Δ1.2.PSD-95:GFP. Very little is localized in the axon (arrows). (D) Δ1.2PSD-95:GFP (green, PDZ1 and PDZ2 deleted) is localized to dendritic spines but not to the axon (arrows). (E) Kv1.4 (red) is localized throughout the cell when cotransfected with GFP.
Neuron  , DOI: ( /S (00) )