Western Blot Analysis of Astrocyte Proteins

Total protein from primary astrocyte cultures was isolated as previously described (Stary et al., 2017 (link)). Briefly, cultures were first washed with cold 0.1% phosphate buffered saline, then total cellular protein was quantified by Pierce BCA protein assay kit [ThermoFisher Scientific (Stary et al., 2017 (link))]. Equal amounts of protein were loaded and separated on 10–12.5% polyacrylamide gels, then transferred to Immobilon polyvinylidene fluoride membranes (EMD Millipore Corp). Membranes were blocked with 5% skimmed dry milk and incubated overnight with primary antibody against SIRT1 (Abcam, #ab110304), MFN2 (Abcam, #ab124773), β-actin (LI-COR Bioscience #926–42,210) and/or β-tubulin (Abcam, #ab6046). Membranes were then washed and incubated with secondary antibodies (LI-COR Bioscience) for 1 h followed by washing again and visualizing by using the LICOR Odyssey infrared imaging system. Densitometric analysis of bands was performed via Image Studio Lite (LI-COR Biosciences), and the intensity of all proteins was normalized to β-actin or β-tubulin as a control.