Sc 377037

After separation of the protein bands by SDS-PAGE, the gel was fixed in 100% MeOH. The gel was then immersed in transfer buffer (tris-hydroxyaminomethane 25 mM, glycine 192 mM, 20% MeOH) and the proteins in the gel were transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer system at a constant current of 180 mA for 60 min. The transferred membranes were fixed and stained with Ponceau stain and then decolorized. Blocking was then carried out using 10% skim milk powder dissolved in TBS-T for 60 min at room temperature. Then, primary antibodies anti-prohibitin 1 (1:100, sc-377037, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-prohibitin 2 (1:100, sc-133094; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-ANT2 (1:1000, ANT2/SLC25A5(E2B9D), Cell Signaling Technology, Danvers, MA, USA), diluted in PBS-T solution containing 1% skim milk, were allowed to react overnight at 4 °C. Shaking and washing with PBS-T for 5 min was performed three times; then, the antibodies were reacted with secondary antibodies for 3 h at room temperature. After 3 rounds of shaking and washing with PBS-T for 5 min, protein detection was performed using SuperSignal West Pico Chemiluminescent Substrate or Super Signal West Femto Maximum Sensitivity Substrate (Bio-Rad, Hercules, CA, USA). An LAS-3000 (FUJIFILM, Tokyo, Japan) was used to photograph the membrane.