Anti parp 1 h 250

Anti-GR (rabbit, H300, Santa cruz Biotechnology), anti-PPP2CA (rabbit, Proteintech), anti-Phosphorylated S211 Glucocorticoid receptor (rabbit, cell signaling technology), anti-STRN3 [1) N-20, goat polyclonal, sc:16853, Santa cruz Biotechnology, used in immunofluorescence experiments, 2) S68, mouse monoclonal, MAI-46461, Thermo Scientific, 3) mouse monoclonal, 05-1115, Millipore] (1 and 2 successfully detect over-expressed STRN3 in western blots), anti-Flag (mouse, Sigma), anti-Flag (rabbit, Sigma), anti-Lamin A/C (mouse, Cell signaling technology), anti-GAPDH (rabbit, Abcam), anti-PARP-1 (H-250, Santa cruz Biotechnology), horseradish peroxidase-conjugated anti-mouse and anti-rabbit from Jackson Immunoresearch laboratories, Pierce donkey anti-rabbit secondary antibody dylight 800 or 680 and Pierce donkey anti-mouse secondary antibody dylight 680 (Thermo Scientific).

Cell lysates were prepared with the ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Burlington, Canada). Twenty µg of the cell lysate isolated from Rin-5F or islet cells or 20 µg proteins precipitated from the cell culture medium after cell treatment were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblot analysis was performed using anti-PARP-1 (H-250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PAR (H10; Alexis Biochemicals, San Diego, CA, USA), anti-Akt 1/2/3 (H-136; Santa Cruz Biotechnology), anti-pAkt 1/2/3 (Ser-473-R; Santa Cruz Biotechnology), anti-ERK 1/2 (K-23; Santa Cruz Biotechnology), anti-pERK (E-4; Santa Cruz Biotechnology) antibodies. Blots were probed with horseradish peroxidase-conjugated secondary antibodies. Staining was performed by the chemiluminescent technique according to the manufacturer's instructions (Amersham Pharmacia Biotech, Amersham, UK). If different set of samples were run on different gels, immunoblot analysis was performed in the same time, under the same conditions. All membranes were exposed under the same film and thus exposure and development did not influence the obtained results.

For Western blot analysis, cells were lysed as previously described [43 (link)-44 (link)]. Protein determination was performed by using the BCA Protein Assay (Thermo Scientific, Rockford, IL). Samples were supplemented with loading buffer (250 mM Tris pH 6.8, 2% SDS, 10% glycerin, 4% beta-mercaptoethanol, 1% bromophenol blue) and boiled for 2 minutes. Equal amounts of protein for each sample were migrated in SDS-polyacrylamide gels and blotted onto nitrocellulose filters, as previously described [43 (link),44 (link)]. The following Abs were used: anti-p53 (DO-1), anti-MDM2 (SMP14), anti-p21 (C-19), anti-PUMAα/β (H-136), anti-PARP-1 (H-250) and anti-Bax (2D2) purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Phospho-p53 (Ser15) and anti-Phospho-p53 (Ser392) from Cell Signaling Technology (Danvers, MA); anti-tubulin from Sigma-Aldrich. After incubation with anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary Abs (Sigma-Aldrich), specific reactions were revealed with the ECL Lightning detection kit (Perkin Elmer, Waltham, MA). Densitometry values for Western blot were estimated by the ImageQuant TL software (GE Healthcare, Buckinghamshire, UK) and were expressed as arbitrary units (a.u.). Multiple film exposures were used to verify the linearity of the samples analyzed and to avoid saturation of the film.