2.8. Western Blotting

Protein levels of PKC-δ, p-Akt, Caspase-3, Bax, and Bcl-XL were detected by western blotting. Brain tissues containing the frontoparietal cortex and dorsolateral striatum were processed as described previously [34] and incubated with primary antibodies (polyclonal rabbit anti-PKC-δ at 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA; polyclonal rabbit anti-phospho-Akt 1:1000, Cell Signaling Technology, Danvers, MA, USA; rabbit polyclonal anti-caspase-3 antibody, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA; rabbit polyclonal anti-cleaved caspase-3 antibody, 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA; rabbit polyclonal anti-BAX antibody, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA; mouse monoclonal anti-Bcl-XL, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C. Western blot images were analyzed by an image analysis program (ImageJ 1.42, National Institutes of Health, Bethesda, MD, USA) to quantify protein expression in terms of relative image density.