Panel A: Western blot showing bands for CAST, ELKS, and Actin proteins under different shRNA conditions. Panel B: Fluorescence images showing surface synaptotagmin-1 and synaptophysin in control and CAST/ELKS-KD neurons. Dot plots quantify surface synaptotagmin-1 fluorescence intensity. Panel C: Fluorescence images showing co-accumulation of ALFA-mCherry-tagged CAST or ELKS with various EGFP-tagged endocytosis-related proteins. Panel D: Fluorescence images showing EGFP-RIM1 interaction with ALFA-mCherry-CAST and ALFA-mCherry-Endophilin-A2.
Surface level of endogenous SV protein in CAST/ELKS-KD neurons and a screening analysis of co-condensation between CAST/ELKS and endocytosis-related proteins in the HEK cells. (A) CAST, ELKS, and tubulin were detected by western blot analysis in rat hippocampal neurons expressing control-shRNA, CAST-shRNA-#1, CAST-shRNA-#2, ELKS-shRNA-#1, ELKS-shRNA-#2, or CAST/ELKS-shRNAs. CAST/ELKS double-knockdown (CAST/ELKS-KD) neurons were generated by co-expression of CAST-shRNA-#1, CAST-shRNA-#2, ELKS-shRNA-#1, and ELKS-shRNA-#2. (B) Surface levels of endogenous synaptotagmin-1 in CAST/ELKS-KD neurons. The left panels show surface-exposed synaptotagmin-1 (green) and total synaptophysin (magenta) in control neurons (upper panels) and CAST/ELKS-KD neurons (lower panels). Scale bar, 2 μm. The dot plots on the right show quantification of the surface synaptotagmin-1 fluorescence intensity (control and CAST/ELKS-KD; n = 225 puncta from 12 images). P values were calculated using the nonparametric Mann–Whitney test. (C) Screening proteins that of co-accumulate with ALFA-mCherry-CAST or ALFA-mCherry-ELKS (magenta), and EGFP-tagged endocytosis-related proteins including synaptojanin-1, dynamin-1, dynamin-2, dynamin-3, intersectin2, endophilin-A1, endophilin-A2, endophilin-A3, AP2-α, CHC, CLC, amphiphysin, syndapin, and FCHO2 (green). Scale bar: 10 μm. (D) EGFP-RIM1 result in droplet-like condensate with ALFA-mCherry-CAST (middle panel), but not condensate with ALFA-mCherry-endophilin-A2. Scale bar: 10 μm. Source data are available for this figure: SourceData FS1.