Infrared dye tagged secondary antibodies

Extracts of transfected cells were prepared in RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing a protease and phosphatase inhibitor cocktail (Halt, Thermo Scientific, Waltham, MA, USA). The cells were first homogenized by vortexing and left on ice for 15 min. The samples were then centrifuged and a total protein concentration in the supernatant was assessed by Bradford assay. From each sample, 60 µg of total protein was denatured at 95 °C for 5 min in 1X Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and 5% 2-mercaptoethanol. Protein samples were then resolved on a 4–12% Bis–Tris gel (Invitrogen, Carlsbad, CA, USA) in 1X SDS/Tris/Glycine buffer, transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and blocked with 1X Odyssey PBS Blocking Buffer (LI-COR, Lincoln, NE, USA) for 1 h at room temperature. The membrane was then probed with anti-c-Myc (1:1,000) (9E10, Cell Signaling, Danvers, MA, USA) and anti-β-Actin (1:2,000) (ab8227) (Abcam, Cambridge, UK) for normalization at 4 °C for 18 h. After incubation with infrared dye-tagged secondary antibodies (LI-COR, Lincoln, NE, USA), immunoblots were washed thrice in PBS-Tween 20 (0.1%) with a final wash in PBS. Protein bands were visualized on a digital imaging system (Odyssey FC, LI-COR, Lincoln, NE, USA).