Anti hp1α

1 × 10⁶ cells were lysed in 100 μL RIPA buffer [50 mmol/L Tris-HCl (pH = 7.5), 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS] supplemented with NaF, NaVO4 and a protease-inhibitor mixture (Roche). Typically 20 μg of proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore), and blotted with one of the following primary antibodies and then HRP-conjugated secondary antibodies. The quantification of western blot was performed with Image Lab software for ChemiDoc XRS system (Bio-Rad).
Primary antibodies for western blotting include anti-WRN (Santa Cruz Biotechnology, Inc.), anti-Progerin (Santa Cruz Biotechnology, Inc.), anti-P21 (Cell Signaling Technology, Inc.), anti-LAP2β (BD Bioscience, Inc.), anti-HP1α (Cell Signaling Technology, Inc.), anti-Actin (Santa Cruz, Inc.), anti-Lamin B1 (Abcam, Inc.), anti-P16 (BD Bioscience, Inc.), anti-H3K9me3 (Abcam, Inc.), anti-β-Tubulin (Santa Cruz, Inc.).

FOXO1^+/+ and FOXO1^–/– hMSCs cultured in 6-well plates were lysed on ice with 120 μL of radioimmunoprecipitation assay (RIPA) buffer (Invent, IN-WB001) supplemented with protease inhibitor cocktail (EDTA-free) (Roche, 4693159001) and PhosSTOP phosphatase inhibitor (Roche, 4906837001) for 30 min, and then centrifuged at 13,500 ×g for 15 min at 4°C to extract the supernatant. Then, the protein concentration was measured using BCA protein quantification kit (Dingguo Changsheng Biotech, BCA02). Each sample was then subjected to SDS-PAGE separation and electrotransferred to PVDF membrane (Millipore, ISEQ00010). Subsequently, the membranes were blocked with 5% nonfat milk in PBST for 1 h at RT and then incubated with appropriate primary antibodies at 4°C overnight. After several washes, membranes were incubated with the secondary antibodies conjugated with horseradish peroxidase (HRP) at RT for 1 h. Imaging was captured with ChemiDoc XRS + system (Bio-Rad) and the data of protein band intensity were analyzed with ImageJ. The following primary antibodies were used for western blotting: anti-FOXO1 (Cell Signaling Technology, 2880), anti-P16^INK4a (BD Bioscience, 550834), anti-GAPDH (Santa Cruz, sc-365062), anti-HP1α (Cell Signaling Technology, 2616), and anti-HP1γ (Cell Signaling Technology, 2619).