In addition, each FingR was expressed in a punctate pattern that localized with the corresponding endogenous protein (Figures 4E–4H, 4M–4P, and S3). In the presence of siRNA against Gephyrin, the total Gephyrin expressed in processes per cell was reduced by 96% ± 1% (Figure 4Q; n = 10 cells) compared with scrambled siRNA, whereas the staining of GPHN.FingR-GFP was reduced by 98.1% ± 1% (n = 10 cells) under the same circumstances, a difference that

is not significant (p > 0.13, t test). Similarly, in cells Z-VAD-FMK in vivo where the amount of total PSD-95 was reduced by 96% ± 1% (Figure 4R; n = 10 cells), the amount of PSD95.FingR staining in processes was reduced by 99% ± 1% (n = 10 cells) compared with cells expressing scrambled siRNA, a difference that is not significant (p > 0.1, t test). These results are consistent with the majority of PSD95.FingR and GPHN.FingR labeling their target proteins within dissociated cortical neurons. In the CNS there are three close homologs of PSD-95 that are also found at postsynaptic selleck sites: PSD-93,

SAP-97, and SAP-102 (Brenman et al., 1998). To determine whether PSD95.FingR could distinguish between different MAGUK proteins, we independently tested whether PSD95.FingR-GFP bound to PSD-93, SAP-102, or SAP-97 in our COS cell assay. We found that PSD-93 did not colocalize with PSD95.FingR-GFP, whereas SAP-102 and SAP-97 did (Figures 5A–5C). To determine whether SAP-102 and SAP-97 interact with PSD95.FingR-GFP in a more stringent assay, we coexpressed HA-tagged versions of these proteins in cultured cortical neurons where PSD-95 expression had been knocked down with siRNA. We found that when PSD95.FingR is coexpressed with either SAP-97 or SAP-102, the coexpressed proteins colocalize (Figures 5D–5K). Thus, PSD95.FingR probably binds to heterologous SAP-97 and SAP-102 with relatively high affinity, but not to heterologous PSD-93. Additional testing will be required to determine the exact specificity of binding of PSD95.FingR-GFP with endogenous MAGUK proteins in vivo. However, even in the case where PSD95.FingR does identify other synaptic MAGUK proteins, it is still

suitable for marking synapses. In addition to testing the specificity Suplatast tosilate of binding, we asked whether expression of the FingR had a morphological effect on cells. In light of the dramatic increase in spine size and density caused by overexpression of PSD-95 (El-Husseini et al., 2000; Kanaani et al., 2002) and the large aggregates seen with overexpression of Gephyrin (Yu et al., 2007), we tested whether expression of PSD95.FingR-GFP or GPHN.FingR-GFP had an effect on the size of PSD-95 or Gephyrin puncta, respectively. We found that the amounts of total Gephyrin associated with individual puncta (stained with an anti-Gephyrin antibody) were nearly identically distributed in cells expressing GPHN.FingR-GFP (μGPHN = 18.1 ± 0.7 a.u., n = 200 puncta) versus with untransfected cells (μGPHN = 18.4 ± 0.8 a.u., n = 200, p > 0.