Pab240

Proteins from cell lysates (50–80 μg of total protein) were fractionated by SDS-PAGE and transferred for 1 h at 200 mA to PVDF membranes (Immobilon; Millipore) using a wet transfer system. The PVDF membranes were blocked for 1 h at room temperature in 5% nonfat milk in TBS-T (TBS with 0.1% Tween), and incubated with primary antibody at an appropriate dilution at 4 °C overnight in blocking buffer. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies diluted in TBS-T buffer for 1 h at room temperature. Immunolabeled proteins were visualized by ECL (General Electric Healthcare, Amersham, UK). Antibodies used for Western blotting were as follows: anti-angiogenin (1:500, ab10600; Abcam), anti-p53 (1:500, PAb240; Abcam), anti-p21 (1:500, sc-6246; Santa Cruz Biotechnology), anti-β-actin (1:10000, C4; Santa Cruz Biotechnology), and anti-SALL2 (1:1000, HPA004162; SIGMA).

Soluble whole-cell lysates of the hMBs treated with 30 μM of iSN04 or AS1411 in DM for 48 h were prepared as described above. The lysates were denatured with 50 mM Tris-HCl (pH 6.8), 10% glycerol, and 2% SDS at 95°C for 5 min. Ten microgram of protein samples were subjected to SDS-PAGE on a 10% polyacrylamide gel followed by Western blotting using an iBlot 2 Dry Blotting System (Thermo Fisher Scientific). 1.0 μg/ml each of rabbit polyclonal anti-nucleolin antibody, mouse monoclonal anti-p53 antibody (PAb 240; Abcam), and mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (5A12; Wako) were used as primary antibodies. 0.1 μg/ml each of horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG antibodies (Jackson ImmunoResearch) were used as secondary antibodies, respectively. HRP activity was detected using ECL Prime reagents (GE Healthcare) and ImageQuant LAS 500. The quantities of nucleolin and p53 proteins were normalized to that of GAPDH using ImageJ software.

Western blot analysis was performed on total proteins extracted from brains of mice irradiated at E11 and dissected 2 h after irradiation. Proteins were harvested by lysing brain tissues with 200 µl RIPA buffer (50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) containing protease inhibitor and phosphatase inhibitors cocktail tablets (Roche, Brussels, Belgium). Western blotting was performed using standard procedures with the following primary antibodies: p53-Ser15-P (catalog number 9284, Cell Signaling Technology, Leiden, The Netherlands), total p53 (Pab 240, Abcam, Cambridge, UK), and Gapdh (ab8245, Abcam, Cambridge, UK) as a loading control. For visualization we used chemiluminescence (Clarity Western ECL Substrate, Bio-Rad, Temse, Belgium).

NP40 lysates of control, Foldlin or MG-132 treated cells (M7449, Sigma Aldrich) were subjected to immunoprecipitation by adding the cell lysate to magnetic beads (Dynabead protein G, 1004D, Life technologies) coated with pAB240 (Abcam, 1 μg/IP reaction) or pAB1620 (Abcam, 1 µg/IP reaction) and incubated overnight at 4 °C. The beads were subsequently extensively washed in lysis buffer, and SDS/Western blot analysis was carried out, as previously described [9 (link)].

Cells grown on glass coverslips were fixed with a 4% paraformaldehyde (PFA) for 15 minutes or 100% methanol for 10 minutes. Primary antibodies were used as follows: goat anti-TAp63 (Santa Cruz, sc-8608, diluted 1:50), rabbit anti-Ki67 (Abcam, ab15580, diluted 1:200), goat anti-ΔNp63 (Santa Cruz, sc-8609, diluted 1:100), rabbit anti-MDM2 (Abcam, ab58530, diluted 1:100), mouse monoclonal anti-Krt18 (Abcam, ab668, diluted 1:100), mouse monoclonal anti-p53 (Abcam, ab26, diluted 1:100), mouse monoclonal anti-p53 (Abcam, PAb 240, diluted 1:100), mouse anti-human BRCA1 (Calbiochem, OP92, diluted 1:100), and mouse monoclonal anti-Nucleophosmin (Invitrogen, FC-61991, diluted 1:200, a kind gift of Karita Peltonen and Prof. Marikki Laiho). Secondary antibodies were donkey anti-goat IgG conjugated with Texas Red (Invitrogen, diluted 1:1000), or goat anti-rabbit IgG conjugated with Alexa Fluor 594 (Invitrogen, diluted 1:1000), or goat anti-mouse IgG conjugated with Alexa Fluor 488 (Invitrogen, diluted 1:1000). Images were taken with a Nikon Eclipse 90i fluorescence microscope and processed using Nikon NIS-Elements AR software.

The cells were seeded on a coverslip (Corning Life Sciences, United States) and then incubated at 37°C overnight. Subsequently the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. The cell membrane was permealized with 0.5% Triton X-100 for 5 min to allow the entry of antibody into cells. Then the permealized cells were blocked with 1% BSA (bovine serum albumin) for 1 h at room temperature, followed by incubation with the primary antibody PAB1620 or PAB240 (Abcam, United States) at 4°C overnight. These antibodies can recognize the p53 protein status. PAB1620 and PAB240 are able to specifically recognize wild-type and mutated p53, respectively (Yu et al., 2012 (link)). Thereafter, the cells were incubated with the goat anti-mouse IgG Alexa Fluor 488 secondary antibody (Invitrogen, United States) for 1 h at room temperature. The nuclei were stained with DAPI (4′, 6-diamidino-2- phenylindole) for 10 min at room temperature. After washes with PBS, the immunofluorescence of cells was detected by laser scanning confocal microscope LSM700 (Zeiss, Germany).