Figure 2: Shisa6 co-localizes with AMPARs and PSD-95 at postsynaptic sites of hippocampal neurons.

(a) Triple-immunofluorescence staining of a cultured Shisa6 KO neuron (DIV16), expressing inducible Flag-tagged Shisa6 for 18 h, for surface expressed Flag-Shisa6 (green), endogenous surface GluA2 (blue) and endogenous PSD-95 (red) shown as a three-channel overlay. (b) Single-channel images and colour overlay of the dendrite region boxed in a. An individual synaptic spine (boxed area) is enlarged and is shown (bottom left inset). (c) Arrows on overlay image of dendrite segment shown in b (left) represent locations of four line scans used to derive graphs shown (right) and illustrate the co-enrichment of Flag-Shisa6, GluA2 and PSD-95 immunofluorescence intensities at synaptic sites. (d) Biochemical fractionation (homogenate (H), crude synaptic membranes (P2; with and without microsomes (M)), synaptosomes (SS), synaptic membranes (SM) and PSD fraction (Triton-X100 insoluble fraction) of mature mouse hippocampus reveals an enrichment of Shisa6 in the PSD together with GluA2, GluN2A (NR2A), PSD-95, and distinct from the presynaptic marker synaptophysin (Syp). (e) Immunoblot analysis of native hippocampal immunoprecipitated GluA2 complexes reveals the co-precipitation of Shisa6 (upper panel). Immunoblot analysis of immunoprecipitated Shisa6 complexes confirms the interaction with GluA2, and identifies GluA1 and GluA3 as additional interaction partners (lower panel). No signal was obtained in the Shisa6 KO. The input controls represent 3% of the total lysate. (f) Flag-Shisa6 (∼61 kDa) binds directly to homomeric GluA1, GluA2 and GluA3 receptors, while having minimal affinity for GluK2, as shown by co-precipitation from HEK293 cells, using a Flag antibody. The input controls represent 2% of the total lysate. For complete blots, in addition to those with higher exposure, see Supplementary Fig. 9.