Western Blot Analysis of Apoptosis Proteins

As previously stated, RIPA buffer containing proteinase inhibitors was utilized to extract total protein from N2a cells or rat brain cortex [23 (link)]. A BCA assay was applied to measure the lysed protein concentrations. Then, proteins from individual samples prepared above were separated by 10% SDS–PAGE and transferred to PVDF membranes overnight at 4 °C. Then, the membrane was probed with appropriate primary antibodies, namely, DOCK4 (CST; 1:500), Bcl2, Bax, caspase3, and GAPDH (Abcam; 1:1000). Blots were subsequently probed using secondary HRP-conjugated goat anti-rabbit IgG antibodies (Abcam; 1:2000) for approximately 90 min at ambient temperature. An improved chemiluminescence kit (Pierce, Thermo Fisher Scientific, Inc.) was employed to detect proteins.