Rabbit anti p21

For Immunofluorescence, 6 μm frozen sections of aortic roots were fixed and permeabilized with cold-acetone for 5 min and blocked in PBS containing 3% BSA for 1 hour at room temperature. Sections were stained with rat anti-CD31 (BD Pharmingen, 550274, 1:30), rabbit anti-p16 (1:100, ab51243, Abcam), rabbit anti-p21 (1:400, 2947, Cell Signaling), rabbit anti-γH2AX (2577, 1:800, Cell Signaling), cleaved caspase3 (1:400, 9661, Cell Signaling), Importin-α3 (1:100, ab84706, Abcam) and rabbit anti-MEKK3 (Cell Signaling) in PBS at 4 oC overnight. After three washes with PBS, the sections were further incubated with Alexa Flour 488-conjugated donkey anti-Rat IgG (Invitrogen, A21208, 1:300) and Alexa Flour 555-conjugated goat anti Rabbit IgG (Invitrogen, 1:300) in PBS for 1 hour at room temperature. Nuclei were stained for 5min at room temperature in PBS containing DAPI (Cell signaling, 4083, 0.5μg/ml). Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen). Images were acquired on an upright Carl Zeiss LSM 510 confocal. The data were calculated from at least 40 cells for each group with 6–10 mice. For each mouse, 2–5 plaques were selected and used for quantification. Tunnel protocol was performed as described by manufacturer’s protocol (Millipore, ApopTaq Peroxidase Detection kit).

Samples were lysed in RIPA lysis buffer (Thermo Scientific) supplemented with a protease inhibitor (Roche). Protein concentrations were measured using the Bradford Assay (Bio-Rad). Equal amounts of total protein were separated by SDS-PAGE and transferred to PVDF membranes, which were then blocked in 5% nonfat milk and incubated overnight at 4°C in blocking buffer containing primary antibodies. Antibodies used for immunoblotting were: rabbit anti-LC3B (1:1000; Sigma), rabbit anti-p21 (1:1000; Cell Signaling), rabbit anti-p27 (1:1000; Cell Signaling), rabbit anti-cyclin D1 (1:1000; Cell Signaling), mouse anti-p62 (1:1000; Abcam), rabbit anti-ERα (1:1000; Cell Signaling), rabbit anti-Atg7 (1:1000; Cell Signaling), rabbit anti-Beclin1 (1:1000; Cell Signaling), rabbit anti-ILK1 (1:100; Cell Signaling), and rabbit anti-GAPDH (1:2000; Cell Signaling). After three washes with TBST, blots were probed with horseradish peroxidase-conjugated anti-rabbit secondary antibodies for 45 min (1:5000; Cell Signaling). Following incubation with ECL Plus Western Blotting Detection System (GE Healthcare), blots were imaged using a FluorChemE Imager (Cell Biosciences). For each protein, densitometry analysis was carried out in Image J by dividing the total intensity of each band by that of GAPDH in the same sample.