Western Blot Analysis of Protein Expression

In order to perform expression studies, protein was extracted from the patients’ and controls’ LCLs as well as the transfected SH-SY5Y cells as described previously (Ververis et al., 2020 (link)). After running in an SDS-PAGE polyacrylamide gel, proteins were transferred to Hybond-P hydrophobic polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). Membranes were blocked for 3 h (LCLs) or 1 h (SH-SY5Y) in 5% non-fat dry milk in phosphate buffered saline (PBS)/Tween, at room temperature, followed by overnight incubation at 4°C with the primary antibodies for the protein of interest and a housekeeping control [rabbit anti-SPG7 (Sigma Aldrich Taufkirchen, Germany)/1:6,500 for LCLs, rabbit anti-GFP (Proteintech, Illinois, United States)/1:5,000 for SH-SY5Y, mouse anti-β-ACTIN (Sigma-Aldrich)/1:4,000 for LCLs, mouse anti-vinculin (7F9) (Santa Cruz Biotechnology, Heidelberg, Germany)/1:1,000 for SH-SY5Y]. The next day membranes were washed 3 × 10 min in PBS/Tween and incubated with the appropriate secondary antibodies for 1 hour. Three washes followed and proteins signals were visualised using the LumiSensor Chemiluminescent HRP Substrate Kit (Genscript, New Jersey, United States), in a UVP BioSpectrum Imaging System (UVP, California, United States). Three independent experiments were carried out for each case. The ImageJ 1.51j8 software was used for data analysis.