Fig. 6: Characterization of SPHKAP and VAP proteins in neurons from Akap11-cKO and Atg7-cKO mouse brains.
a IF imaging of the hippocampal CA2, CA3, and DG of the brains slices from Akap11F/F and Akap11F/F; Syn-cre mice, stained with anti-SPHKAP and DAPI. Inset images provide magnified views of cortex, hippocampus, thalamus, and striatum. Scale bars, 200 μm (main images); 5 μm (inset images). Bar graph as the quantification of SPHKAP puncta numbers and average size in DAPI-positive cells using Analyze Particles in ImageJ. Data are presented as mean ± SEM. For puncta numbers: CA2: n = 5 for Akap11F/F and n = 4 for Akap11F/F; Syn-cre; CA3: n = 6 and n = 5; DG: n = 9 and n = 10. For average puncta size: CA2: n = 5 for Akap11F/F and n = 5 for Akap11F/F; Syn-cre; CA3: n = 6 and n = 6; DG: n = 10 and n = 10. Statistical significance was performed using an unpaired two-tailed t-test (p < 0.0001). Each experiment was independently repeated three times with similar results. b Volcano plot of the differentially expressed proteins (DEPs) detected by mass-spectrometry in Atg7F/F and Atg7F/F; Syn-cre mouse brains. A positive score indicates enrichment, a negative score indicates depletion. The y axis represents statistical confidence FDR for each x axis point. Enriched proteins, defined by FDR < 0.05 and |Log2FC | > 2 SD, are colored in red. FDR was calculated with the moderated two-sided t-test in the LIMMA package for multiple comparisons. c IF imaging of the cortex from Atg7F/F and Atg7F/F; Syn-cre mice, stained with anti-SPHKAP and DAPI. Scale bars, 5 μm. Bar graph as the quantification of SPHKAP puncta numbers per DAPI-positive cell using Analyze Particles in ImageJ. Data are presented as mean ± SEM (Atg7F/F: n = 17; Atg7F/F; Syn-cre: n = 13). Statistical significance was performed using an unpaired two-tailed t-test (p < 0.0001). Each experiment was independently repeated three times with similar results. d, e, f IF imaging of the cortex from Akap11F/F and Akap11F/F; Syn-cre mice, stained with anti-SPHKAP, anti-VAPA, anti-RIβ, and DAPI. Scale bars, 5 μm. Bar graph as the quantification of SPHKAP-VAPA (e) puncta numbers and (f) average size in DAPI-positive cells using Analyze Particles in ImageJ. Data are presented as mean ± SEM. For puncta numbers (e): Akap11F/F, n = 7; Akap11F/F; Syn-cre, n = 4 (p = 0.0310). For average size (f): Akap11F/F, n = 7; Akap11F/F; Syn-cre, n = 4 (p = 0.0005). Statistical significance was performed using an unpaired two-tailed t-test. Each experiment was independently repeated three times with similar results. g IF imaging of the cortex from Akap11F/F and Akap11F/F; Syn-cre mice, stained with anti-SPHKAP, anti-VAPA, anti-p62, and DAPI. Each experiment was independently repeated three times with similar results. Scale bars, 5 μm. h Co-immunoprecipitation was performed using anti-HA antibody from HEK293T cells expressing HA-tagged AKAP11 WT or FFAT motif mutant (mFFAT), followed by immunoblotting with antibodies against HA, AKAP11, RIα, RIβ, VAPA, and VAPB. Each experiment was independently repeated three times with similar results. i HEK293T cells expressing HA-AKAP11 WT or mFFAT were stained with antibodies against HA, VAPA, and DAPI. The bar graph shows quantification of HA–VAPA co-localized signal using Manders’ coefficient. Data are presented as mean ± SEM (WT: n = 6; mFFAT: n = 9). Statistical significance was performed by an unpaired two-tailed t-test (p < 0.0001). Each experiment was independently repeated three times with similar results. Scale bars, 5 μm. j Primary neurons expressing HA-AKAP11 WT or mFFAT were stained with antibodies against HA, VAPA, and MAP2. The bar graph shows quantification of HA–VAPA co-localized signal using Manders’ coefficient. Data are presented as mean ± SEM (WT: n = 4; mFFAT: n = 5). Statistical significance (p = 0.0245) was performed using an unpaired two-tailed t-test. Each experiment was independently repeated three times with similar results. Scale bars, 5 μm.
