Anti annexin 5

GTL-16 cells were lysed in iced Complete Tablet Buffer (Roche) supplemented with 2 mM sodium orthovanadate and 35 μg of proteins resolved on 15 or 5 % SDS-PAGE gels. Polyvinylidene fluoride membranes (PVDF, GE Healthcare Europe GmbH, Milan, Italy) were incubated overnight at 4 °C with specific primary antibody: anti-phospho-ERK^{Thr202/Tyr204} (1:1000, Cell-SIgnaling), anti-VDR (1:250, Santa-Cruz), anti-Annexin V (1:1000, Sigma), anti-Beclin1 (1:250, Santa-Cruz), anti-Caspase 8 (1:400, Sigma), anti-Bax (1:200, Santa-Cruz), anti-Vitronectin (1:250, Santa-Cruz) and anti-Fibronectin (1:250, Abcam, UK). The protein expressions were normalized and verified through β-actin detection (1:5000; Sigma, Milan, Italy).

Caco-2 and GTL-16 cells were lysed in ice Complete Tablet Buffer (Roche, Milan, Italy) supplemented with 2 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich), 1:50 mix Phosphatase Inhibitor Cocktail (Sigma-Aldrich) and 1:200 mix Protease Inhibitor Cocktail (Calbiochem, San Diego, CA, USA) and 35 μg of proteins of each sample were resolved on 8% and 15% SDS-PAGE gels. Polyvinylidene difluoride membranes (PVDF, GE, Healthcare Europe GmbH, Milan, Italy) were incubated overnight at 4 °C with specific primary antibody: anti-annexin V (1:2000; Sigma-Aldrich), anti-p53 (1:250, Santa Cruz Biotechnology, Heidelberg Germany), anti-ferroportin (1:250, Santa Cruz Biotechnology), anti-ferritin (1:250, Santa Cruz Biotechnology) and anti-DMT1 (1:250, Santa Cruz Biotechnology). Protein expression was normalized and verified through β-actin detection (1:5000; Sigma-Aldrich) and expressed as mean ± SD (% vs. control).