Anti mcl 1

Caco-2 or LS174T cells were seeded into 10 cm petri-dish (2 × 10⁶) and treated with thiazolides (20 μM), cisplatin (10 μg/ml), DMSO (0.1%), or with complete medium as control for 0, 2, 4, and 6 h and additional 8 h. In some experiments, Caco-2 cells were pretreated with JNK inhibitor (2.5 μM) or with p38 inhibitor (10 μM) or with NAC (10 mM) for 1 h. before treatment with RM4819, cisplatin, or DMSO for up to 16 h. Cells were then lysed in NP40-lysis buffer (150 mM NaCl, 50 mM Tris pH 7.6, 1 mM EDTA and 1% NP-40) and cell lysates were separated on a denaturing 12% SDS-PAGE gel. After transfer to polyvinylidene difluoride membranes (PVDF) (Roche, Mannheim, Germany), Mcl-1, Bcl-x_L, Bim, Puma, phospho-JNK, phospho-p38, or tubulin as loading control were detected using specific antibodies (anti-Mcl-1 from eBioscience (Frankfurt, Germany); anti-Bcl-x_L (54H6), anti phospho-SAPK/JNK (Thr183/Tyr185) (81E11), anti-phopho–p38 MAPK (Thr180/Tyr182) (D3F9), anti-Puma (D30C10) from Cell Signaling Technology (Davers, MA, USA); anti-Bim, anti-α-Tubulin from Sigma-Aldrich). Proteins were detected using horse radish-coupled secondary antibodies and enhanced chemiluminescence on an Image Quant LAS 4000 (GE Healthcare, Braunschweig, Germany).