Histone 2b

MES23.5 cells were treated with P7C3 for 2 h. After washing, the cells were treated with MPP⁺ for 24 h. The cells were then collected, and cytosolic and mitochondrial fractions were isolated using the Mitochondria Isolation Kit for cultured cells (Beyotime). For immunoblot analysis, TOM20 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-Tubulin (Abcam, Cambridge, UK) were selected as the markers for mitochondria and cytosol, respectively. For obtaining the nuclear and cytoplasmic fractions, cells were treated as described above and then lysed in the fractionation buffer containing 3 mM CaCl₂, 2 mM MgAc, 320 mM sucrose, 0.1 mM EDTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5% NP-40 for 20 min on ice. After centrifugation for 15 min at 600 × g at 4 °C, the supernatant was collected as the cytoplasmic fraction. The pellets were washed twice with the fractionation buffer without NP-40 and lysed in the nuclear lysis buffer containing 280 mM KCl, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20 mM Hepes (pH 7.9), 25% glycerol, 1.5 mM MgCl₂, and 0.3% NP-40 as the nuclear fraction. Histone 2B (Abcam) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, Billerica, MA, USA) were selected, respectively, as the markers for nucleus and cytoplasm in immunoblot analyses.