Protein Extraction and Analysis

After treatment for 24 hours (RI and ME) or seven days (MB), cells were harvested and total protein was extracted by using RIPA buffer with protease inhibitor. Total protein concentration was quantified by Bio-Rad DC Protein Assay (Bio-Rad). Equal amount of protein extract was loaded and separated by electrophoresis in 9% SDS-PAGE gel. Proteins were transferred onto a PVDF membrane and probed with primary antibodies mEM48 and γ-tubulin (Millipore) at 4°C overnight followed by thorough washes and incubation with secondary antibody. Positive band was visualized by using SuperSignal West FemtoChemiluminescent Substrate (Thermo Scientific) and imaged by ChemiDoc Imaging System (Bio-Rad).^{4 (link)}