Anti p53 sc6243

Immunoblotting and co-immunoprecipitations were performed as described [48 (link)]. Antiserum against FOXO (C29H4) and anti-Flag M2 (F3165) was purchased from Cell signaling Technology and Sigma for immunoblot analysis. E2F1 antibody (C20) was purchased from Sigma. Anti-HA antibody was purchased from Roche (3 F10). Anti-p53 (sc-6243, Santa Cruz), p-FOXO1 (9461S, Cell Signaling Technology), p-Akt (sc-7985-R, Santa Cruz), Capase-3 (Cell Signaling Technology, Cat# 9661), Ki-67 (SP6) (Biocare Medical, Cat# CRM325), and TUNEL (EMD Millipore, Cat# S7101) were used for IHC. IHC detection was through primary antibodies listed in Additional file 1: Table S2, with Vector biotinylated secondary (1:250), tertiary was streptavidin-horseradish peroxidase (Covance #SIG-32000) and chromagen 3,3′-diaminobenzidine substrate (Covance #SIG-31043).

The tissue samples were fixed in 10% neutral-buffered formalin within 1 h after surgical removal and paraffin-embedded using standard procedures. Then the fixed paraffin-embedded specimens were cut into 5-μm-thick sections, which were deparaffinized in xylene, rehydrated in ethanol, and microwave treated for antigen retrieval. The appropriately diluted primary antibody, anti-p53 (SC-6243, Santa Cruz Biotechnology) and anti-FOXO3a (#12829, Cell Signaling Technology), were incubated for 60 min at room temperature in a humidity chamber. Slides were then rinsed in PBS and subsequently incubated with the secondary antibody against anti-rabbit antibody and the visualization of HRP (concentration 1:300, #074-1506, KPL). Then the slides were washed and the sections were developed in the enzyme substrate diaminobenzidine (DAB, Sigma) solution. Images of immunohistochemically stained sections were captured by the Olympus digital microscope (IX-73P1F, Olympus, Japan).

Western blot analysis was performed as previously described (Racca et al., 2011 (link)) and the following antibodies were used: rabbit polyclonal anti-H-Ras (C-20, sc-520, Santa Cruz), mouse monoclonal anti-KLF6 [clone 2c11, previously specified by Gehrau et al. (2011) (link)], rabbit and goat polyclonal anti-p53 (sc6243, Santa Cruz Biotechnology), mouse anti- α -tubulin (T9026, Sigma-Aldrich), mouse anti-β-actin (A5316, Sigma-Aldrich) and donkey-anti-mouse near-Infrared fluorescent secondary antibodies 680CW and 800CW (LI-COR Biosciences, Lincoln, NE, United States). α-tubulin and β-actin were used as loading control. Fluorescence emission was acquired with Odyssey CLx scan (LI-COR, United States). Images are representative of three independent experiments.

The protein expression levels of p53, FOXO3a, HuR, RPS19BP1, DBC1 in brain tissues were analyzed using western blotting analysis. The total protein of brain tissues was extracted using RIPA Lysis Buffer (Beyotime, China) according to the manufacturer’s instructions. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, China). 5 mg of proteins were separated using 12% SDS-polyacrylamide gels and transferred onto a PVDF membrane (0.45 μm, Millipore, USA). The blots were blocked with 5% non-fat milk in TBS, then incubated overnight at 4°C with the appropriate dilution of primary antibodies: anti-p53 (SC-6243, Santa Cruz Biotechnology), anti-FOXO3a (#12829, Cell Signaling Technology), anti-HuR (#12582, Cell Signaling Technology), anti-AROS (Ab201091, abcam), anti-DBC1 (#5857, Cell Signaling Technology), anti-β-action (AC004, ABclonal Technology). After washing the membranes to remove excess primary antibody, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibodies at a dilution of 1:2000-1:5000 (AC004 or AS003, ABclonal Tehcnology). The membranes were washed 3 times and then visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific). β-action was used as an internal control.

Protocol to separate cytosolic and nuclear for immunoblotting was as previously reported [47 (link)]. In brief, B cells (1 × 10⁷) were lysed in 20 µl buffer A (10 mM Hepes, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 0.1% Triton X-100, with protease inhibitor freshly added) and left on ice for 5 min. The lysate was centrifuged by 1300 × g, 4 °C for 4 min to receive nuclei pellet. The supernatant was further clarified by 20,000 × g, 4 °C for 15 min to remove cell debris and insoluble aggregates and receive the cytosolic fraction. Nuclei were washed once in buffer A, and then lysed in 30 µl buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and freshly added protease inhibitors). After centrifugation by 1700 × g, 4 °C for 5 min, the yielded supernatant as nuclear fraction was collected. In-house anti-PP4 antibody was generated as described previously [42 (link)]. Antibodies recognizing p-p53 S15 (#9284), p-ATR S428/S431 (#2853), p-Chk1 S345 (#2348), p-NBS1 S343 (#3001), NBS1 (#14956), γH2AX (#9718), or H2AX (#7631) were from Cell Signaling. Antibodies recognizing ATM (2C1), p-ATM S1981/1987 (10H11.E12), GAPDH (GTX100118), or p84 (5E10) were from GeneTex; and anti-p53 (SC-6243) was from Santa Cruz. ECL Plus^TM Western Blotting Detection Reagents (Amersham) were utilized for immunoblot development.

Liver tissues were homogenized and digested in 1× RIPA buffer containing phosphatase inhibitor cocktail solution (GenDEPOT, Barker, TX, USA). Western blot experiments were performed following the standard protocol. The following primary antibodies were used: anti-c-Myc (ab32072; Abcam, Cambridge, UK), anti-p53 (sc-6243; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-GAPDH (#2118; Cell Signaling Technology, Danvers, MA, USA). Anti-rabbit IgG–HRP (Sigma-Aldrich, St. Louis, MO, USA) was used as the secondary antibody. Bands were detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).