P53 sc 6243

Brain tissues were homogenized in a tissue extraction buffer containing 2% SDS and protease inhibitor (Sigma, St. Louis, MO, USA P8340) and phosphatase inhibitor cocktails (Sigma, P5726). The homogenates were centrifuged at 3000 x g, 4°C, for 10 min and then in 100,000 x g for 60 min. Westerns were conducted with supernatants and the following antibodies: PAI-1 (ASMPAI-GF, ASHPAI-GF, Molecular Innovation, Novi, MI, USA), GFAP (Cat No G9269, Sigma-Aldrich, Inc., St. Louis, MO USA), p53 (SC-6243, Santa Cruz), p21 (SC-397, Santa Cruz, Dallas, TX, USA) and GAPDH (G9545, Sigma). The protein bands were visualized using the ECL detection system (Amersham, Piscataway, NY, USA), semi-quantified by ImageJ software. The results were normalized with GAPDH, a housekeeping gene.

Cells were harvested and washed twice with PBS. Cell lysis was performed on ice for 25 min, in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl, pH 8.0) containing a protease inhibitory cocktail (Roche). Insoluble material was pelleted by centrifugation at 16,000× g at 4°C for 3 min. Protein concentrations were determined using the Bradford assay (Bio-Rad). Thirty micrograms extract was mixed with 4× Laemmli buffer (40% glycerol, 240 mM Tris/HCl, pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% β-mercaptoethanol), denatured at 96ºC for 5 minutes, separated by SDS-PAGE, and transferred to nitrocellulose membranes (PROTRAN-Whatman, Schleicher&Schuell). The membranes were blocked with 5% non-fat dry milk in TBS-T for 60 min, incubated with primary antibodies overnight at 4°C, washed three times with TBS-T for 10 min, incubated with the peroxidase-conjugated secondary antibody (1:2000; Amersham Biosciences) in TBS-T with 5% non-fat dry milk for 60 min, and washed three times with TBST for 10 min. Immunoreactive proteins were detected using Supersignal West Dura HRP Detection kits (Pierce). The primary antibodies used were: p16^Ink4a (Santa Cruz); p19^Arf (Ab80 Abcam); p15^Ink4b (Santa Cruz); β-catenin (clone 14, BD Biosciences); p53 (sc-6243 Santa Cruz); p21 (BD Pharmigen); c-Myc (sc-764 Santa Cruz); β-actin (ab8226, abcam).

Whole cell extracts were either separated on a 7% Tris-Acetate polyacrylamide (PA) gel (HUWE1, BRCA1) or on a 10% Tris-Glycine PA gel and transferred to an Immobilon-FL membrane (Millipore) for subsequent immunoblotting. Primary antibodies were detected using infrared (IR) Dye-conjugated secondary antibodies (Rockland). The signal was visualized using direct IR fluorescence via the Odyssey Scanner, LI-COR Biosciences. Primary antibodies: BRCA1 (sc-6954, Santa Cruz), Cdc6 (ab155759, Abcam), c-Myc (sc-789, Santa Cruz), HDAC2 (sc-9959, Santa Cruz), HUWE1 (A300-486A, Bethyl Laboratories), p53 (sc-6243, Santa Cruz), Polλ (A301-640A-1, Bethyl Laboratories), TopBP1 (NB100-217, Novus Biologicals), Tubulin (T9026, Sigma-Aldrich). All original images of immunoblots are depicted in Supplementary Fig. S1.

Protein extracts were electrophoresed on polyacrylamide gel (Invitrogen) and transferred onto nitrocellulose membranes (Millipore). After 1 hour (h) blocking with 5% dry fat milk in phosphate-buffered saline (PBS) containing 0.02% Tween-20, the membranes were incubated with the primary antibody overnight at 4 C° and with the secondary antibody for 1h at room temperature. Primary antibodies used are: anti-human BARD1 (cod-A300-263A, Bethyl, 1:1000), γH2AX (phosphoSer139) (cod-H5912, Sigma Aldrich, 1:1000), phosphor-p53(Ser-15) (cod-9284 Cell Signaling, 1:500), p53 (sc-6243, Santa Cruz, 1:500), Cyclin B (sc-752 Santa Cruz, 1:500), CDK1 (sc-54, Santa Cruz, 1:1000); phospho-H3 (06-570 Millipore). Mouse monoclonal anti-β-Actin antibody (cod-A5441, Sigma-Aldrich, 1:6000) and anti-H3 (cod-06-755, Millipore, 1:1000), were used as loading control for cytosol and nuclei extracts respectively. Secondary peroxidase-labeled antibody to rabbit IgG (cod041506, KPL) and to mouse IgG (cod041806, KPL) were diluted at 1:2000. Protein bands were visualized with enhanced chemiluminescence plus reagent (GE Healthcare). The protein bands image were acquired with GelDoc 2000 system (Bio-Rad) and the densitometry measurement was performed by Quantity One 4.5 tool (Bio-Rad).

Lysates of cells and harvested liver tissues were prepared using RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing protease inhibitor (Sigma-Aldrich), phosphatase inhibitor cocktail 2 (Sigma-Aldrich), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich). Total protein concentration of the lysates was measured using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Proteins were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA), and probed with appropriate dilutions of the following antibodies: p53 (sc-6243, 1:1000, Santa Cruz), p21 (ab109199, 1:1000, Abcam, Cambridge, MA, USA), PAI-1 (sc-5297, 1:1000, Santa Cruz), TTP (T5327, 1:2000, Sigma-Aldrich), and α-tubulin (2125S, 1:1000, cell signaling, Danvers, MA, USA). Then, membranes were incubated with secondary antibodies (115-035-003; HRP-Goat Anti-Mouse IgG, 111-035-003; HRP-Goat Anti-Rabbit IgG) at room temperature for 30 min. Antibody binding was visualized with an ECL chemiluminescence system (Pierce Biotechnology) and chemiluminescence signal was read by Azure Biosystems C300 analyzer (Azure Biosystems, Dublin, CA). The relative band density was analyzed by using ImageJ2x software (US National Institutes of Health, Bethesda, USA).