Pab421

Cell surface proteins were analyzed by a FACScan flow cytometer and the Lysis II software (BD Biosciences, Heidelberg, Germany). Briefly, 10⁶ cells were incubated with the specific primary antibody detecting B7-1 (clone 16-10A1), B7-2 (clone GL-1), H-2K[d] (SF1-1.1), H-2D[d] (clone 34-2-12), or I-A[d] (AMS-32.1) in 20 μl of phosphate buffered saline. After 45 min cells were washed and incubated with a fluorescein isothiocyanate (FITC) conjugated secondary antibody for 30 min. Antibody concentrations were used as indicated by the manufacturer (BD Biosciences). Free antibodies were removed by washing and samples were analyzed by flow cytometry in 500 μl of phosphate buffered saline.
For detection of intracellular p53, cells were suspended in 1 ml RPMI 1640 medium containing 50% fetal calf serum and fixed for 30 min on ice by drop wise addition of 3 ml 100% ethanol at −20°C. Then cells were washed with 3 × 10 ml phosphate buffered saline before an anti-p53 antibody (PAb 421, Millipore, Schwalbach, Germany) and a FITC conjugated secondary antibody (BD Biosciences) were added according to the recommendations of the manufacturer. Flow cytometry was performed in a volume of 500 μl phosphate buffered saline, after removing unbound antibodies by washing.

The anti-p53 antibodies Pab421, Pab246, CM5 and 1C12 were purchased from Millipore (Darmstadt, Germany), Vector laboratories (Burlingame, CA, USA) and Cell signaling (Danvers, MA, USA), respectively. From Santa Cruz (Dallas, TX, USA), we obtained the antibodies against Oct-4 (C-10) Nanog (C-4), c-Jun (H79) and PCNA (PC10). Antibodies against acetylated and phosphorylated p53 (K379, S6, S15, S392) were from Cell signaling. Antibodies against GAPDH (6C5), PARC (PO69) and α-7 (MCP72) were from Hytest (Turku, Finland), BioLegend (San Diego, CA, USA) and Enzo (Loerrach, Germany), respectively. Antibodies against β-Actin and Histone H3 were purchased from Abcam (Cambridge, UK) and the antibody against MdmX (MDMX82) was from Sigma-Aldrich.
siRNAs were purchased from Eurofins (Ebersberg, Germany). Sequences are available on request. siRNA transfections were performed using Macsfectin (Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer's instructions.

HDQ-P1, H1299-R213X, and empty vector-transfected H1299 cells were seeded in six-well plates with glass slides, at a density of 300,000 cells per well for HDQ-P1 cells and 200,000 cells per well for H1299 cells. The next day, cells were treated with 100 µM G418. After 72 h treatment, cells were washed in PBS, fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton, and washed in PBS. Anti-p53 antibody PAb421 (Merck, Darmstadt, Germany) was diluted 100 times in 2% BSA and anti-p53 antibody FL393 (Santa Cruz, CA, USA) was diluted 400 times in 2% BSA. Both antibodies were mixed and incubated with the cells for 2 h at room temperature. After washing with PBS, cells were incubated with secondary antibodies conjugated with Alexa Fluor dye (Invitrogen/Thermo Fisher Scientific, Sweden), for 1 h at room temperature, washed with PBS and mounted with HardSet mounting medium with DAPI (Vector Laboratories, CA, USA). Images were obtained with a Zeiss Axioplan 2 microscope with an AxioCam HRm Camera.