Hybond p hydrophobic polyvinylidene difluoride pvdf membranes

Proteins from each sample were loaded and separated onto SDS-PAGE 12% polyacrylamide gels and were transferred to Hybond-P hydrophobic polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). Membranes were blocked for 1 hour at room temperature and were incubated overnight at 4°C with the respective specific primary antibody for each protein [mouse anti-LRSAM1/Abcam ab73113 (1:400), rabbit anti-LRSAM1/Novus Biological H00090678-D01 (1:750), mouse anti-TSG101/Novus Biological NB200-11 (1:500) and mouse anti-β-ACTIN/Sigma-Aldrich A2228 (1:4000)]. Then, incubation with the appropriate secondary antibodies was performed for 2 hours and followed by incubation with the visualization LumiSensor Chemiluminescent HRP Substrate Kit (Genscript, U.S.A.) at room temperature. Membranes were visualized using the UVP imaging system (BioRad, U.S.A.). Quantification of Western blots was performed using the ImageJ program (https://imagej.nih.gov). The protein quantity ratio was estimated relative to β-Actin.

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.