Western blotting

and p53 conformational immunoprecipitation Total cell extracts were prepared by incubation in lysis GSK458 datasheet buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 150 mM KCl, 1 mM dithiothreitol, 1% Nonidet P-40) and a mix of protease inhibitors and resolved by 9-12% SDS-polyacrilamide gel LY411575 electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Millipore) and membranes were blocked with 5% nonfat dry milk in PBS and incubated with the primary antibodies followed by an anti-immunoglobulin–G-horseradish peroxidase antibody (BioRad). Immunoblotting was performed with the following antibodies: monoclonal anti-poly(ADP-ribose) polymerase (PARP, BD Pharmingen, CA, USA), monoclonal anti-p53 (Ab-DO1), polyclonal anti-p53 (FL393) and polyclonal anti-Bax (all from Santa Cruz Biotechnology), purified mouse

anti-phospho-Histone H2AX (Ser139) (Millipore, clone JBW301; kindly provided by S. Soddu, JIB04 Regina Elena National cancer Institute, Rome, Italy) and monoclonal anti-β-actin (Calbiochem). Enzymatic signals were visualized by chemoluminescence (ECL kit, Amersham Corporation). P53 protein conformation was evaluated essentially as described [9]. Briefly, cells were lysed in immunoprecipitation buffer (10 mM Tris, pH 7.6; 140 mM NaCl; 0.5% NP40, and protease inhibitors) for 20 min on ice, and cleared by centrifugation. Pre-cleared supernatants (200 μg) were immunoprecipitated overnight at 4°C with the conformation-specific monoclonal antibodies Pab1620 (wild-type specific) and PAb240 (mutant specific) (Calbiochem) [18, 19] pre-adsorbed to protein G-agarose (Pierce). Immunocomplexes were collected by centrifugation, separated by 9% SDS-PAGE and blotted onto PVDF membrane (Millipore). Immunoblotting was performed with rabbit polyclonal anti-p53 (FL393). Immunofluorescence staining The cells were grown on coverslips and treated with Zn-curc (100 μM) for 24 h. After treatment, cells were fixed in 4% formaldehyde for 10 min and then premeabilized with 0.5% Triton X-100 for 5 min before staining

with conformation antibodies PAb1620 and PAb240 at 1:200 dilution in PBST, overnight at 4°. Erastin Cells were then visualized on a Nikon Eclipse Ti-U fluorescence microscope (Nikon) and the percentage of fluorescent cells was assayed by scoring 200 cells/field, three times and normalized to Hoechst staining. RNA extraction and semi-quantitative reverse transcription (RT)-PCR analysis Cells and glioblastoma tissues were harvested in TRIzol Reagent (Invitrogen) and total RNA was isolated following the manufacturer’s instructions essentially as described [20]. PCR was performed by using genes specific oligonucleotides under conditions of linear amplification. PCR products were run on a 2% agarose gel and visualized by ethidium bromide staining using UV light. The housekeeping β-actin mRNA was used as internal control.