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.