Panel A shows a western blot analysis with four lanes for different protein samples. The lanes are labeled with various protein markers: Anti-CAST, Anti-Tubulin, Anti-Endophilin-A1, and Anti-Endophilin-A2. The molecular weight markers are indicated on the left side. Panel B consists of immunofluorescence images showing the localization of CAST and Bassoon in hippocampal neurons. The images are divided into four rows: CAST (endogenous), 3x Flag-CAST WT, 3x Flag-CAST 7RKA+CR, and 3x Flag-CAST 7RKA+6R9KA. Each row contains three columns: CAST, Bassoon, and Merge. The right side of Panel B features a dot plot showing the Pearson correlation coefficient between CAST and Bassoon across different conditions. Panel C displays STED microscopy images of CAST and Bassoon in CAST/ELKS-cDKO mouse hippocampal neurons expressing either wild-type or mutant CAST. The images are divided into two rows: CAST-wt and CAST-mt. Each row contains three columns: CAST, Bassoon, and Merge. Arrows indicate sites of co-localization. The right panels show mean fluorescence intensity profiles from 10 active zones.
The interaction with CAST/ELKS with endophilin is required for the stimulation-evoked SV response. (A) CAST-mutants expression level in mouse hippocampal neurons from CAST-flox/ELKS-flox mouse. CAST and tubulin were detected by the western blot analysis in control (Cre-mutant), CAST/ELKS-cDKO (Cre), CAST/ELKS-cDKO with 3xFlag-CAST-WT (Cre with CaMKII0.3 promoter-derived CAST), with 3xFlag-CAST-7RKA+ΔCR, and with 3xFlag-CAST-7RKA+6R9KA. (B) The localization of CAST-mutants in the hippocampal culture neurons. Control, CAST/ELKS-cDKO, CAST/ELKS-cDKO with CAST, or CAST/ELKS-cDKO with CASTmt (CAST-7RKA+ 6R9KA) neurons are stained with CAST and Bassoon. Scale bar: 2 µm. Right dot plots show the correlation co-efficiency between CAST vs Bassoon (n = 10 images, each). P values are compared with the cDKO with CAST (one-way ANOVA with post hoc Bonferroni’s multiple comparison test). (C) Immunofluorescence images acquired by STED microscopy showing CAST (magenta; WT in the top, mutant [7RKA+6R9KA]) and Bassoon (orange) in CAST/ELKS-cDKO mouse hippocampal neurons expressing either WT or mutant CAST. Arrows indicate sites of co-localization of CAST and Bassoon. Scale bars: 1 µm. The right panels show mean fluorescence intensity profiles from 10 active zones. Source data are available for this figure: SourceData FS3.