Western blotting

Whole-cell lysates were collected 48 h post-transfection by treatment with lysis buffer (100 mm Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.2% Triton X-100, 1% PMSF, protease inhibitor cocktail; all from Sigma–Aldrich). Cells were lysed for 10 min at 4 °C followed by centrifugation for 30 min at 10,000 g at 4 °C. Proteins were resolved on 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad) and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in 1–5% milk for 1 h at room temperature and then probed with mouse-anti-EGFP (for pYFP constructs; 1:8000; Clontech) or rabbit-anti-Rluc antibody (for pRluc constructs; 1:2000; GeneTex) for 2 h at room temperature. Next, membranes were incubated with HRP-conjugated goat-anti-mouse (1:2000; Bio-Rad) or donkey-anti-rabbit antibody (1:2000; Abcam) for 1 h at room temperature. Bands were visualized with Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) using a ChemiDoc XRS + System (Bio-Rad). Equal protein loading was confirmed by stripping and reprobing with mouse-anti-β-actin antibody (1:10,000; Sigma).