(a) Shisa7 is closely related to the AMPAR auxiliary subunit Shisa6 and Shisa9, bearing a signal peptide (SP; 22 amino acids), an extracellular domain with conserved cysteine-rich motif, a single transmembrane region (TM), and an intracellular domain with a type II PDZ-ligand motif (EVTV). Exon4 (Ex) is an alternative-splice region in Shisa7 and Shisa9, whereas this is Exon3 in Shisa6. The predicted molecular weight of the two mature Shisa7 protein variants is ~56 and ~54 kDa, and that of Shisa6 ~59 and~55 kDa (Klaassen et al., 2016). (b) Shisa7 is highly enriched in the cortex, olfactory bulb and hippocampus, and absent in cerebellum, as measured in crude synaptic membrane fractions. Despite the presence of both transcript variants (see Figure 1—figure supplement 1), the indicated protein band (~68 kDa; arrow head) is dominant in the hippocampus. Lower panel depicts the loading control, that is, total crude synaptic membrane protein. (c) Immunohistochemistry of primary Shisa7 KO hippocampal neurons (DIV14) after viral overexpression of Flag-Shisa7 shows Shisa7 expression (green) in endogenous Homer1-positive puncta (red). The lower panels show a zoom-in (white box in upper panel). The overlay of the two channels is shown, scale bars are indicated. (d) Biochemical fractionation (homogenate (H), crude synaptic membranes (P2; with and without microsomes (M)), synaptosomes (SS), synaptic membranes (SM) and postsynaptic density fraction (PSD; Triton X-100 insoluble fraction) of mature mouse hippocampus reveals an enrichment of Shisa7 in the PSD together with GluA2, GluN2B, PSD-95, and this pattern is distinct from the presynaptic marker Synaptophysin (Syp). (e) Precipitation of Flag-Shisa7 (~60 kDa) from HEK293 cells using a Flag antibody shows that upon co-expression it binds directly to homomeric GluA1, GluA2 and GluA3 receptors, whereas having minimal affinity for GluK2. For complete blots, in addition to those with higher exposure, see Figure 1—figure supplement 3. The input controls represent 2% of the total lysate.