P53 antibody do 1

Immunoblot analysis was carried out as previously described.^{18 (link)} Briefly, 4–20 × 10⁶ cells were treated with different reagents as indicated and lysed in RIPA buffer (50 mM Tris HCl, 137 mM NaCl, 0.5% Na Deoxycholate, 1% Triton X-100, 0.1% SDS, protease inhibitors). Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences, Little Chalfon, UK) using a semi-dry blotting approach. The following antibodies were used: p53 (DO-1) antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Tubulin and actin (A5441) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA).

H1299 cells were seeded (200 000 cells per well) into a six-well plate (Falcon) and 24 h later co-transfected—in the presence of the Jet Pei reagent (Ozyme), as recommended by the manufacturer—with pCMVp53WT or its mutant derivatives (3 μg), variants of p53BS-luc (2 μg) and pCMLacZ (0.6 μg). Media was exchanged six hours after transfection. The antibiotic G418 (200 μg/ml) was added with the fresh media 6 h post-transfection. Protein extracts were prepared 24 h after transfection and enzymatic activities were measured as previously described (46 (link)). For each experimental set-up, at least five independent transfection experiments were performed. For visualization of short and long forms of the p53 protein, protein extracts were prepared using 200 μl of a room tempered 1× Glo buffer; incubation for 5 min at 25°C while shaking at 550 rpm. Approximately 0.5 μg of protein extracts were subjected to the ‘Western-like’ analyses using the Jess Protein Simple device; the total protein was measured by Bradford. The p53 antibody (DO-1; the N-terminal epitope mapping between amino acid residues 11–25 of p53; Santa Cruz Biotechnology) was used at its saturating conditions 1:100. The equal loading was monitored by the total protein staining (Protein Simple).

Co-IP assay was carried out as described previously with modifications.^{43 (link)} H1299 cells were transfected with different cDNA expressing p53-72P, p53-72R or MDM2 for 24 h, and treated with HL001 (20 μM) for 36 h. Cells were washed with cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH=7.0, 150 mM NaCl, 1 mM EDTA and 1% NP-40 supplemented with protease inhibitor cocktail). The whole-cell lysates were incubated with 2 μg p53 antibody DO-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 20 μl protein A/G Sepharose beads (Santa Cruz Biotechnology) overnight at 4 °C. The immunocomplexes were washed with lysis buffer for three times and separated by SDS–PAGE followed by western blotting analysis. As p53 protein band is overlapping with IgG bands, IgG light chain was further detected by using specific anti-IgG light chain secondary antibody from (Jackson immunoResearch, West Grove, PA, USA).