2.11. Analysis of MFN‐1 and PINK1 protein expression in BMSCs

In order to determine the extracellular level of accumulated proteins, the cell cultures were lysed by the use of ice‐cold RIPA buffer supplemented with 1% of protease and phosphatase inhibitor cocktail (Sigma‐Aldrich, Munich, Germany). The Bicinchoninic Acid Assay Kit was used to determine the amount of isolated protein (Sigma‐Aldrich, Munich, Germany). The samples containing 8 µg of protein were mixed with 4× Laemmli loading buffer and incubated at 95°C for 5 min in T100 Thermal Cycler (Bio‐Rad, Hercules, CA, USA). The electrophoresis reaction (SDS‐PAGE) was performed in 11% sodium dodecyl sulphate‐polyacrylamide gel for 90 minutes at 100V using Mini‐PROTEAN Tetra Vertical Electrophoresis Cell (Bio‐Rad, Hercules, CA, USA). Subsequently, the samples were transferred into polyvinylidene difluoride membrane (PVDF) using the Mini Trans‐Blot® system (Bio‐Rad, Hercules, CA, USA) for 1h at 100V. Then, membranes were blocked by the use of 5% skim milk powder in TBST buffer for 1h and then incubated overnight at 4°C with primary antibodies. The used primary antibodies were anti MFN‐1 antibody produced in rabbit (orb11040, Biorbyt) in dilution 1:500 and anti PINK1 antibody produced in rabbit (orb331223, Biorbyt) in dilution 1:250. The reference was anti β‐ACT antibody produced in rabbit (a5441, Sigma‐Aldrich) in dilution 1:2500. Membranes were washed 5 times for 5 min in TBST buffer. The incubation with secondary antibodies was performed for 1h at 4°C (Goat Anti‐Rabbit IgG Antibody in dilution 1:2500, ap156p, Sigma‐Aldrich). Subsequently, membranes were washed 5 times as described previously and analysed using Bio‐Rad ChemiDoc™ XRS system (Bio‐Rad, Hercules, CA, USA). The chemiluminescent signal was detected by the use of DuoLuX^® Chemiluminescent and Fluorescent Peroxidase (HRP) Substrate (Vector Laboratories). The signal intensity and molecular weight of detected proteins was analysed using Image Lab™ Software (Bio‐Rad, Hercules, CA, USA).