Immunoblotting for Protein Quantification

The protein levels in cells were determined by immunoblotting as previously mentioned^{23 (link)}. Briefly, cells were washed with ice cold PBS and lysed in MPER lysis buffer (Thermo Fischer Scientific) containing protease inhibitors cocktail (Thermo Fischer Scientific) for 15 min on ice. The cell debris was removed by centrifuge at 10000 × g for 15 min at 4 °C. The protein levels in clear cell lysate were measured by Bradford assay (VWR Life Science). Total protein (40 μg) was resolved by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked in 5% skim milk for 45 min at room temperature followed by probing with 1:1000 diluted primary antibodies (1:250 for ORP5) overnight at 4 °C. After three washes with TBST (50 mM Tris-Cl, 150 mM NaCl, and 0.1% Tween 20; pH7.6), membrane was incubated with respective HRP-conjugated secondary antibodies (1:1000) for 2 h at room temperature and washed three times. The luminescent signal was developed either using Super Signal West Femto Maximum Sensitivity or Dura Extended Duration substrate (Thermo Fischer Scientific) and images were captured using Gel Doc system (Bio-Rad). Image Lab software (Bio-Rad) was used for densitometry of bands.