2.9. Western blot
The extraction of total protein was performed by lysing the cells in Lysis buffer: 1% NP40, 50 mM HEPES [pH 7], 150 mM NaCl, 1 mM EDTA, supplemented with protease inhibitors. The protein samples (20 μg per well) were resolved by electrophoresis in TGX Stain Free Precast gels (Bio-Rad, Spain), each sample run in triplicate, and UV trans-illumination (1 min) images were obtained to control for loading (Bio-Rad ChemiDoc MP Imaging System, Spain). The proteins were then transferred to a PVDF membrane and after blocking for 30 min in 5% BSA, the membranes were probed overnight at 4 ºC with a mouse anti-β-actin antibody (1:200,000 dilution: Sigma (A1978), Japan). The membranes were washed thrice with 0.01% Triton-X 100 in PBS and incubated for 1 hour with the secondary antibody (1:10,000 dilution: ThermoFisher 926-32210, Spain). After washing, images were obtained on an Odyssey CLx Infrared Imaging System (Li-Cor, USA) and the protein bands were quantified with ImageJ software (USA). The β-actin levels were normalized to the total protein level.