Western blot

Cells were harvested and lysed with RIPA buffer (Cell signaling, 9806) in ice for 30 min and centrifuged at 12000× g for 10 min. Supernatant was collected and protein concentration was measured using BCA method (Thermo Fisher Scientific). Protein samples were boiled in 1x NuPAGE LDS sample buffer (Invitrogen) at 70℃ for 10 min. Twenty microgram protein samples were subjected to 7.5% - 15% gradient SDS-PAGE gel and transferred to PVDF membrane (MACHEREY-NAGEL, Germany). The membranes were blocked in 1x Roti-Block (Carlroth, Germany) at room temperature for 1 h and were then probed with specific primary antibodies overnight at 4°C. Blots were incubated with HRP-conjugated secondary antibody (Invitrogen, 31430 and 31460) for 1 h at room temperature. Bands were visualized by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and detected using the ChemoStar ECL Imager (Intas Science Imaging, Germany).