Fig. 2: SPHKAP-type I PKA closely associates with Kv2.1 and VAPs.

Total internal reflection fluorescence (TIRF) images and ground-state depletion (GSD) reconstruction maps of cultured neurons immunolabeled for SPHKAP and a Kv2.1, c RI, or e VAPA/B (TIRF scale bars, 5 µm); representative of n = 5 [Kv2.1], n = 7 [RI], and n = 5 [VAPA/B] cells. Two magnified regions marked by boxes are shown for each panel; x-y fluorescence intensity profile from region indicated by dotted line show areas of proximity between SPHKAP and a Kv2.1 (scale bars i & ii = 1 µm), c RI (scale bar i = 1 µm; scale bar ii = 500 nm), and e VAPA/B (scale bar i = 1 µm; scale bar ii = 500 nm). b, d, f Histograms of nearest neighbor distances to SPHKAP centroids for b Kv2.1 (n = 4863 particles/3 cells), d RI (n = 3972 particles/4 cells), or f VAPA/B (n = 7140 particles/3 cells) centroids. Histograms were fit with a b Gaussian or d, f sum of two Gaussians function; mean values of distributions and R² values depicted on graphs. g Representative images of proximity ligation assay (PLA) puncta (magenta) in neurons expressing scrambled shRNA (scr) or one of two distinct anti-SPHKAP shRNAs (A or B) and immunolabled for Kv2.1 and SPHKAP. The neuron periphery is outlined in each image; scale bar, 10 µm. Mean ± s.e.m PLA signal density is presented below each image. Each point represents 1 cell; n = 31 (scr), 12 (A), 16 (B), 12 (Kv2.1 ab alone), and 11 (SPHKAP ab alone) cells; statistical significance determined using one-way ANOVA followed by Tukey’s multiple comparisons test (vs. scr); ****P < 0.0001, ***P = 0.0001, **P = 0.0004, *P = 0.0009. h As in g, but for SPHKAP-VAPA/B PLA signal density. Each point represents 1 cell; n = 19 (scr), 15 (A), 16 (B), 11 (SPHKAP ab alone), and 12 (VAP ab alone) cells; statistical significance determined using one-way ANOVA followed by Tukey’s multiple comparisons test (vs. scr); ****P < 0.0001. Source data are provided as a Source Data file.