24 well plate

Human embryonic kidney (HEK-293) cells (ATCC) were plated on 50 µg/ml poly-L-lysine (Sigma Aldrich Inc.)-coated coverslips (Fisherbrand, Inc.) in 24-well plates (JetBiofil) and maintained in DMEM (Hyclone) containing 10% fetal bovine serum (Hyclone) supplemented with 5 mg/ml penicillin-streptomycin (Hyclone). Cells at 80% confluency were transfected with 0.5 µg of GFP-CASK-WT and GFP-CASK^L209P DNA per well using the calcium phosphate method. Twenty hours post-transfection, cells were washed twice with phosphate buffered saline (Sigma Inc.) and fixed for 15 minutes at room temperature using a 4% paraformaldehyde solution. Coverslips were mounted on microscope slides (Premiere) using Vectashield (Vector Laboratories Inc.) and visualized using confocal laser scanning microscopy (ZEISS Axio Examiner.Z1 LSM 710). The percentage of transfected cells with visible aggregates was counted using the cell counter plugin of the Image J program. For each condition, five high-power field images were analyzed for aggregation. Total cells and cells containing aggregates were visually identified and tallied. Percent of total cells containing aggregates was then calculated. The image analysis procedure was repeated 3 times for each condition and averaged.

HEK293 cells were plated on 50μg/ml poly-L-lysine (Sigma Aldrich Inc.)-coated coverslips (12-545-80 microscope coverglass; Fisherbrand, Inc.) in 24-well plates (JetBiofil) and co-transfected with 0.25 μg EGFP-CASK DNA in the pEGFP-C3 vector, 0.25 μg FLAG-tagged neurexin-1β DNA in the pCMV vector and 0.25 μg ds-RED-liprin-α3 DNA using calcium phosphate [19 (link)]. Two days post-transfection, the cells were washed in PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. Cells were permeabilized using PBS with 0.01% Triton-X 100 solution and blocked using 5% fetal bovine serum. Cells were immunostained using monoclonal anti-FLAG antibody (1:100) followed by Alexa-633(1:250) anti-mouse antibody. Coverslips were mounted on microscope slides (Premiere) using Vectashield (Vector Laboratories Inc.) and visualized using confocal laser scanning microscopy (ZEISS Axio Examiner.Z1 LSM 710) at room temperature with sequential scanning. Images were acquired using Zeiss ZEN 2011 acquisition software. Colocalization was quantified by using the Image Correlation Analysis plugin of ImageJ. Values of Mander’s Overlap coefficient (R) were reported as percent colocalization.

Human embryonic kidney (HEK-293) cells (ATCC) were plated on 50 µg/ml poly-L-lysine (Sigma Aldrich Inc.)-coated coverslips (Fisherbrand, Inc.) in 24-well plates (JetBiofil) and maintained in DMEM (Hyclone) containing 10% fetal bovine serum (Hyclone) supplemented with 5 mg/ml penicillin-streptomycin (Hyclone). Cells at 80% confluency were co-transfected with 0.5 µg of FLAG-tagged neurexin1-β and GFP-CASK-WT or GFP-CASK^L209P DNA per well using the calcium phosphate method. Twenty hours post-transfection, cells were washed twice with phosphate buffered saline (Sigma Inc.) and fixed for 15 minutes at room temperature using a 4% paraformaldehyde solution. Cells were permeabilized using PBS with 0.01% Triton-X 100 solution and blocked using 5% fetal bovine serum. Cells were immunostained using monoclonal anti-FLAG antibody (1:100) followed by Alexa-633(1:250) anti-mouse antibody. Mounting and imaging procedures are described above.