Western Blotting for Protein Analysis

Western blotting was performed as previously described^{53 (link)}. Tissues and cells were homogenized in ice-cold RIPA buffer containing protease inhibitor (cOmplete Protease Inhibitor Cocktail, Merck) and phosphatase inhibitor (PhosSTOP, Sigma). Supernatants were collected after centrifugation and mixed with an equal amount of loading buffer (125 mM Tris–HCl pH 6.8, 30% glycerol, 10% SDS, and 0.6 M DTT). After heat denaturation, the protein samples were subjected to SDS-PAGE using 10% acrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against phospho-p44/42 MAPK (ERK1/2) (Thy202/Thy204) (9101S; Cell Signaling Technology, Danvers, MA, USA), p44/42 MAPK (ERK1/2) (9102S, Cell Signaling), and actin (MA5-11869; Thermo Fisher Scientific), and then with secondary antibodies against rabbit IgG (7074S; Cell Signaling Technology) and mouse IgG (NA931; GE Healthcare, Little Chalfont, UK). Signals were detected with the Pierce ECL Prime system (GE Healthcare) in a LAS4000 instrument (GE Healthcare). Band densities were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).