Mouse anti p53 do 1

p53 knockdown was achieved as described previously (17 (link)) using validated small interfering RNA (siRNA) (Ambion s605; sense strand, 5′-GUAAUCUACUGGGACGGAATT). The control siRNA was designed, using the siRNA target finder, to have no sequence specificity for any Homo sapiens or Ad5 sequence (Ambion; sense strand, 5′-GAGCCGGACGGCCAAAGAAAUU). Western blot protein detection used the following antibodies at the indicated dilutions: 1:10,000 mouse anti-p53 (DO-1; Santa Cruz), 1:10,000 rabbit anti-Ad late proteins (AdJLB1) (8 (link)), 1:500 rat anti-E4 Orf3 (6A11) (33 (link)), 1:25,000 mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GA1R; Thermo Scientific), 1:10,000 rabbit anti-TFII-I (H58; Santa Cruz), and 1:10,000 mouse anti-FLAG (M2; Sigma). The horseradish peroxidase (HRP)-conjugated secondary antibodies were 1:10,000 goat anti-mouse IgG or goat anti-rat IgG (Sigma) and 1:100,000 goat anti-rabbit IgG (Santa Cruz). Chromatin immunoprecipitation (ChIP) used antibodies to TFII-I as described above and normal rabbit IgG (Santa Cruz).

Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. For detection of proteins, membranes were incubated with antibodies diluted in 5% milk powder in Tris-buffered saline solution containing 0.1% Tween-20. Antibodies against phospho-groups were diluted in 5% BSA instead of milk powder. The following primary antibodies were used: goat anti-G2E3, mouse anti-hsc70, mouse anti-p53 (DO-1) (all Santa Cruz Biotechnology), rabbit anti-Caspase 3, rabbit anti-cleaved Caspase 3, mouse anti-Chk1, rabbit anti-PARP, rabbit anti-phospho-Chk1 (Ser317), rabbit anti-phospho-Chk2 (Thr68) (all Cell Signaling Technology), mouse anti-Chk2, mouse anti-Mdm2 (Ab-1), mouse anti-p21, mouse anti-PARP (all Calbiochem), mouse anti-actin (AC-15), rabbit anti-IgG (all Abcam), mouse anti-phospho-H2AX (Ser319) (Millipore), mouse anti-HA-tag (16B12, Covance), mouse anti-GFP (Clontech). Primary antibodies were detected by peroxidase-coupled secondary antibodies (Jackson ImmunoResearch Europe) using a Chemoluminescence Imaging System (Intas).

Antibodies used in this study include affinity-purified rabbit anti-Mad1 antibodies prepared against amino acids 333–617 of human Mad1^{2 (link)} and diluted 1:3000 for western blots and 1:2000 for immunofluorescence, mouse anti-SUMO1 (21C7, Developmental Studies Hybridoma Bank) diluted 1:250, mouse anti-Myc (9E10, Developmental Studies Hybridoma Bank) diluted 1:200, mouse anti-tubulin (12G10, Developmental Studies Hybridoma Bank) diluted 1:5000, rabbit anti-GFP (#2555S, Cell Signaling), mouse anti-p53 (DO-1, #sc-126, Santa Cruz), rabbit anti-p53 (#NB200-171, Novusbio), mouse anti-actin (JLA20, Developmental Studies Hybridoma Bank) diluted 1:500, rabbit anti-tubulin (#2144S, Cell Signaling), rabbit anti-p21 (#ab188224, Abcam), rabbit anti histone H3 (# 9715S, Cell Signaling), mouse anti-FLAG M2 (Sigma) diluted 1:2500, rabbit anti-PML (sc-5621) diluted 1:500, and mouse anti-HA (#901501, Biolegend), and pan-cytokeratin antibody (AE1 + AE3 conjugated to Alexa Fluor® 647, Novus, cat. no. NBP2-33200AF647).

MYCN3 cells were plated in 96-well plates with coverslip bottoms that were pre-treated with poly-D-lysine. Cells were treated overnight under the treatment conditions described in the main text. Concentrations of the compound used were as follows: 1 μM doxycycline, 5 μM Nutlin, or media control. Cells were washed and fixed using PHEM buffer. Rabbit anti-MYCN (Santa Cruz Biotechnology) and mouse anti-p53 (DO-1, Santa Cruz Biotechnology) primary antibodies were used to detect the interaction between these two proteins using the Duolink In Situ Red kit (Sigma Aldrich) according to the manufacturer’s instructions. Cells were co-stained with DAPI prior to imaging with a Nikon Eclipse Ti microscope. Z-stack images were obtained at 60× magnification, and maximum intensity projections were generated. To quantify PLA spots, size and intensity thresholds were applied to all images, and the number of spots for each cell was scored by attributing the spots to the closest nucleus. Cells with too many spots to count were assigned a value of 125 spots. At least 24 cells were scored per condition.

Western blot assay was performed as previously described [48 (link)]. Antibodies used include: mouse anti-p53 DO-1 (Santa Cruz Biotechnology, Santa Cruz, CA), Rabbit anti-MDM4 (Bethyl Laboratories), mouse anti-BAX (Invitrogen), mouse anti-a-tublin (Santa Cruz) and mouse anti-β-actin (Sigma Aldrich).