Western blotting

Whole cell lysates were prepared from HeLa cell resuspended in RIPA buffer (150 mM NaCl, 1% Triton, 1% Na-Deoxycolate, 0.1% SDS, 50 mM Tris-HCl pH 8.0), supplemented with proteases and phosphatases inhibitors (ROCHE) on ice for 30 min. Proteins were cleared by centrifugation at 20000xg for 10 min at 4 °C. Protein concentration was determined by BCA method. Protein extracts were denatured in 1X Laemmli buffer supplemented with 10 mM dithiothreitol (DTT) at 95 °C for 5 min. For non-denaturating condition DTT was not added and samples were boiled at 75 °C for 5 min. Samples were loaded on SDS-polyacrylamide gels and separated by electrophoresis. Proteins were transferred to 0.2 μm nitrocellulose membranes. Membranes were blocked in 5% fat free milk for 1 hour at room temperature, incubated with primary antibody (α-HA, α-FLAG, α-MCU, α-Parkin) overnight at 4 °C and with secondary antibodies for 1 hour at room temperature. Signal was developed by chemiluminescence, bands were visualized by ECL and quantified by ImageJ software. All of the results are expressed as means ± SEM, and Student’s t test was used for the statistics. α-HA, α-FLAG were from Cell Signaling Technology, α-MCU was from Sigma-Aldrich and α-Parkin from Santa Cruz.