Cytoplasmic and Nuclear Protein Isolation from Heart Tissue

Heart tissues were homogenized in lysis buffer, and protein concentration was determined as described earlier [29 (link)]. Nuclear and cytoplasmic fractions were isolated as described earlier [29 (link)]. PVDF membranes were incubated with TNF-α (1 : 200, Santa Cruz Biotechnology), MIP-2 (1 : 100, Abcam China), tubulin, NFκB p65 antibody, anti-TFEB antibody, LAMP1, ATG5, p62, and histone H3 (1 : 200, Santa Cruz Biotechnology) overnight at 4°C. After three repeated washes, the membranes were probed with corresponding HRP-conjugated secondary antibody (1 : 2000, Rockland, Gilbertsville) for 1 h at room temperature. After three repeated washes, the membranes were detected by chemiluminescence and were exposed on an X-ray film for autoradiography.