Imaging Collybistin Localization in HEK293 Cells

These were performed essentially as previously described (Long et al., 2016 (link)). HEK293 cells were co-transfected with the pRK5myc-hCB3_SH3-^R356Q construct at a 1:1 ratio with pEGFP-gephyrin using electroporation (Gene Pulser II, Bio-Rad). Cells were fixed after 24 h for 2 min in 4% (w/v) PFA in PBS. Immunostaining to detect collybistin was performed using a mouse anti-c-myc antibody (1:200, Sigma) and detected using an AlexaFluor 546 goat anti-mouse secondary antibody (1:600; Invitrogen). Counterstaining for cell nuclei was performed with DAPI (1:500; Life Technologies). Confocal microscopy was performed using a Zeiss LSM 710 META. All images were taken with a ×63 objective.