Anti cleaved caspase 7 asp198

Western blot analysis was carried out as described (Corno et al., 2017 (link)). Briefly, samples were fractionated by SDS-PAGE and blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried no fat milk, and then incubated overnight at 4°C with the following antibodies: anti-phospho-Akt (Ser473), anti-Akt (BD Science, Franklin Lakes, NJ, United States), anti-phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187), anti-ERK1/2, anti-AR (Millipore, Burlington, MA, United States); anti-Hsp90 (ac-Lys294) (Novus, Centennial, Colorado, United States), anti-Hsp90 (Santa Cruz Biotechnology, Dallas, TX, United States), anti-acetylated alfa-tubulin (Sigma-Aldrich, Milan, Italy), anti-Bax and anti-FLIP_L (Sigma-Aldrich, Milan, Italy), anti-p53 (Dako, Santa Clara, CA, United States), anti-cleaved caspase-3 (Asp175) and anti-cleaved caspase-7 (Asp198) (Cell Signaling, Danvers, MA, United States). Anti-vinculin (Sigma-Aldrich, Milan, Italy), anti-β-tubulin (Abcam, Cambridge, United Kingdom) or anti-actin (Sigma) antibodies were used as control for loading. Antibody binding to blots was detected by chemo-luminescence (Amersham Biosciences, Cologno Monzese, Italy). Three independent experiments were performed.

Whole cell lysates were prepared using a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing a mixture of protease and phosphatase inhibitors. Total proteins were subjected to SDS-PAGE, and the antibodies (Abs) used for the study were as follows: anti-ET_AR and anti-β-actin (C-11), purchased from Abcam (UK) and Santa Cruz Biotechnology (CA, USA), respectively; anti-p-p44/42 MAPK (Thr202/Tyr204), anti-p44/42 MAPK, anti-pAkt (S473), anti-Akt, anti-cleaved-PARP (Asp214), and anti-cleaved-caspase 7 (Asp198), were from Cell Signaling Technology (MA, USA). Anti-E-cadherin and anti-N-cadherin were purchased from BD Biosciences (CA, USA). Western blotting filters were developed using the ECL-plus detection system or, otherwise, the SuperSignal West Femto kit (Thermo Scientific, IL, USA). Western blots for ET_AR were quantified by densitometric analysis using ImageJ software.