*2.5. Western Blot Analysis*

Western blot was performed as described previously, with some modifications [24,25]. The proteins were loaded and separated by SDS–PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-PSQ, Transfer membrane, Merck Millipore, Burlington, MA, USA). The immunoreaction was carried out for 16 h at 4 ◦C using an appropriate ratio of the primary antibodies. After that, the membranes were washed with 1× TBST three times for 10 min, and reacted with a horseradish peroxidase-conjugated secondary antibody for

2 h, as appropriate. The expression of the respective proteins was detected using an ECL (Enhanced chemiluminescence) -detection reagent, according to the manufacturer's instructions. The expressions of the different proteins were obtained on X-ray films and were scanned, and the optical densities of the bands were analyzed by densitometry, using the computer-based ImageJ software (version 1.50, NIH, https://imagej.nih.gov/ij/, Bethesda, MD, USA).
