Ecl lightning detection kit

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.

For Western blotting analysis, cells were lysed as previously described [63 (link), 64 (link)]. Protein determination was performed by BCA Protein Assay (Thermo Scientific, Rockford, IL). Equal amounts of protein for each sample were migrated in SDS-polyacrylamide gels and blotted onto nitrocellulose filters. The following Abs were used: anti-Mcl-1 (S-19) and anti-Bcl-2 (100) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-AMPKα (Thr172, D79.5E), anti-AMPKα, anti-phospho-STAT3 (Ser727) and anti- STAT3 from Cell Signalling (Danvers, MA); anti-phospho-Akt1/PKBα (Ser473) from Merck Millipore (Darmstadt, Germany); anti AKT/PKBα from BD; 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). The estimation of the densitometry values of bands was obtained by the ImageQuant TL software (GE Healthcare, Buckinghamshire, UK).