Fig 2: Frrs1l deficiency leads to changes in AMPA receptor subunit levels with no change in gene expression. (A) qPCR for Gria1-4 shows no changes between Frrs1l-/- and wild-type controls for Gria1 and Gria2, with only slight but significant changes for Gria3 and Gria4 (P<0.05 and P<0.001, respectively). (B,C) GLUA1, GLUA2 (B) and GLUA4 (C) immunoreactivities are all significantly lower in Frrs1l-/- adult cerebellum than in control adult cerebellum (**P<0.01, ***P<0.001). The patterns of GLUA2 and GLUA4 immunoreactivity differ between Frrs1l-/- and wild-type controls, with a diffuse band present for GLUA2 in wild type, but only a thin band in Frrs1l-/-. GLUA4 blots also indicate changes in immunoreactive band mobility. (D-F) Other synaptic proteins – PSD95, SNAP25 (D) and CAMKII (E) and NMDAR (GRIN1) (F) – remain unchanged between Frrs1l-/- and wild-type controls. Data analysed using Student's t-test (n=4 Frrs1l+/+, n=4 Frrs1l-/-). ns, non-significant.

Fig 3: (A) Immunofluorescent pictures of dispersed islet cells isolated from Arfrp1flox/flox and Arfrp1ß-cell-/- mice stained with indicated antibodies, nuclei visualized in blue. Scale bar represents 20 µm. (B) Western Blots of isolated islets from Arfrp1flox/flox and Arfrp1ß-cell-/- mice probed with indicated antibodies. (C) Immunofluorescent pictures of dispersed islet cells isolated from Arfrp1flox/flox and Arfrp1ß-cell-/- mice, infected with control adenovirus (Ad-CMV-Null) or a Snap25-Myc encoding adenovirus (Ad-Snap25-Myc) and stained with indicated antibodies, nuclei visualized in blue. Scale bar represents 20 µm.

Fig 4: Disruption of ARFRP1 dependent scaffold leads to degradation of SNAP25. (A) Immunofluorescent pictures of dispersed islet cells from Arfrp1flox/flox and Arfrp1ß-cell-/- mice stained for SNAP25. Nuclei are visualized by DAPI, scale bar represents 40 µm. (B) Western blot analysis of islets isolated from Arfrp1flox/flox and Arfrp1ß-cell-/- mice probed with indicated antibodies. (C) Quantification of Western blots shown in (B) of 8 mice per genotype. Relative SNAP25 levels normalized to loading control is shown as mean ± SEM. * = p < 0.05 (unpaired students t-test, Welch's correction). (D) Western blot analysis of whole cell lysates from Min6 cells treated with indicated adenoviruses at a MOI of 100 and harvested at indicated time points post-infection. Knockdown efficiency for Arfrp1 is 50% after 48 h, and 60–70% for Gopc after 48 h. Membranes were probed with indicated antibodies. (E) Immunofluorescent pictures of Min6 cells infected with indicated adenoviruses. Nuclei were visualized with DAPI. Scale bar represents 20 µm. (F) Quantification of Western blots from whole cell lysates of Min6 cells treated with indicated adenoviruses and cycloheximide (CHX) for indicated time points in 3 independent experiments. Membranes were probed with indicated antibodies. (G) Western blot analysis of whole cell lysates from Min6 cells treated with indicated adenoviruses and chloroquine (CQ) or solvent control (Co = MiliQ) and harvested 36 h post-infection. Membranes were probed with indicated antibodies.

Fig 5: (A) Co-immunoprecipitation assays from HeLa cells co-expressing SNAP25 with GFP-tagged ARFRP1. Membranes were probed with indicated antibodies. (B) Co-immunoprecipitation assays from HeLa cells co-expressing SNAP25 and GOPC. Membranes were probed with indicated antibodies.