Orb11040

Cells were detached from culture dishes and homogenized in RIPA buffer plus protease inhibitor cocktail. The lysates were centrifuged at 4°C for 20 minutes (14 000 g) and supernatants were transferred to new tubes. Thirty micrograms of protein were used for each sample. SDS‐PAGE was performed at 100 V for 90 minutes in Tris/glycine/SDS buffer. Proteins were transferred onto a polyvinylidene difluoride membrane (Bio‐Rad) using a transfer apparatus at 100 V for 1 hour at 4°C in Tris/glycine buffer. After transfer, the membrane was washed with Tris/NaCl/Tween buffer (TBST) and blocked overnight at 4°C with 5% non‐fat milk in TBST. Next, the membrane was washed with TBST and incubated overnight with primary antibody for: mitofusin 1 (MFN) (orb11040; Biorbyt, Cambridge, UK), PINK (orb331233; Biorbyt), caspase‐3 (437800; Life Technologies), beta‐actin (A5441; Sigma‐Aldrich) and mitochondrial fision factor ‐MFF (orb325479; Biorbyt) at a dilution of 1:500. After washing the membrane, solution of appropriate secondary antibody conjugated with HRP was applied. After 2 hours incubation, the membrane was washed again with TBST and incubated with Luminata Forte substrate (Merck, Darmstadt, Germany) and visualized using chemiluminescence method with ChemidocMP (Bio‐Rad).

Cells were detached from culture dishes and homogenized in RIPA buffer plus protease inhibitor cocktail. The lysates were centrifuged at 4°C for 20 minutes (14 000 g) and supernatants were transferred to new tubes. Thirty micrograms of protein were used for each sample. SDS‐PAGE was performed at 100 V for 90 minutes in Tris/glycine/SDS buffer. Proteins were transferred onto a polyvinylidene difluoride membrane (Bio‐Rad) using a transfer apparatus at 100 V for 1 hour at 4°C in Tris/glycine buffer. After transfer, the membrane was washed with Tris/NaCl/Tween buffer (TBST) and blocked overnight at 4°C with 5% non‐fat milk in TBST. Next, the membrane was washed with TBST and incubated with primary antibody for 2 hours: βAKT (Sigma‐Aldrich, A5441), PARKIN (Novus, NB100‐91921), LAMP‐2 (Abcam, ab15580), TET‐2 (Abcam, ab124297), MFN‐1 (Biorbyt, orb11040,) and MFF (Biorbyt orb325479) at a dilution of 1:500. After washing the membrane, a solution of appropriate secondary antibody conjugated with ALP was applied. After 2‐hours incubation, the membrane was washed again with TBST and incubated with BCIP^®/NBT‐Purple Liquid Substrate for 15 minutes. The reaction was stopped by washing the membrane with water.