Western Blot Analysis Protocol

Western blot analysis was performed essentially as described by Verbrugge et al.^{[22 (link)]}. Briefly, cell lysates were prepared from 5 × 10⁶ cells suspended in 150 µL ice-cold lysis buffer (Cell Signalling Technology, #9803) containing 4% Protease Inhibitor Cocktail (PIC) and 1 mM NaVO₄. Supernatant fractions were collected by centrifugation (13,000 × g for 10 min, 4 ^oC), and 30 μg protein aliquots were resolved on a 4%-20% TGX pre-cast SDS PAGE gels (Bio-Rad), followed by transfer onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) suitable for chemiluminescent detection by the Odyssey Infrared Imaging System (PerkinElmer, Zaventem, Belgium). The membranes were pre-incubated in blocking buffer (Odyssey Blocking Buffer, LI-COR, Biosciences, Nebraska, USA) for 1 hr. Next, membranes were incubated overnight (4 ^oC) with primary antibodies and β-actin for control of equal loading. After three washing steps (PBS/0.05% Tween20), the membranes were incubated (1 hr) with secondary antibodies, followed by antibody detection with the LI-COR Odyssey scanner (Biosciences) and digital image acquisition/quantification with the Odyssey infrared imaging system software (version 3.0.16, LI-COR Biosciences) according to the manufacturer’s instructions.