Ecl prime detection kit

Mouse monoclonal anti-ABCB4 (clone P3-II-26) and anti-MRP2 (multidrug resistance-associated protein 2; clone M2-I-4) antibodies were purchased from Enzo Life Sciences (Villeurbanne, France); and anti-α-tubulin (clone 1E4C11) was sourced from ProteinTech (Manchester, United Kingdom). Alexa Fluor-labeled secondary antibodies, the DRAQ5 fluorescent probe and culture media came from ThermoFisher (Cergy-Pontoise, France), and peroxidase-conjugated secondary antibodies were acquired from Rockland Immunochemicals (Gilbertsville, PA, USA). The ECL-Prime detection kit was from VWR (Courtaboeuf, France). The transfection reagents Turbofect and JetPrime were purchased from Thermo-Fisher Scientific, (Saint-Herblain, France) and Ozyme (Saint-Cyr-l’Ecole, France), respectively. Ivacaftor (VX-770) came from Clinisciences (Nanterre, France). All other reagents were obtained from Sigma-Aldrich (Lyon, France).

The mouse monoclonal P3II-26 anti-ABCB4 antibody was obtained from Enzo Life Sciences (Villeurbanne, France). Rabbit polyclonal anti-MRCKα and anti-MRLC were from Euromedex (Souffelweyersheim, France). Monoclonal anti-myc and anti-α-tubulin antibodies were from ThermoFisher (Cergy-Pontoise, France) and ProteinTech (Manchester, United Kingdom), respectively. The mouse monoclonal anti-GFP was from Roche (Meylan, France). Alexa Fluor-labeled secondary antibodies, DRAQ5 fluorescent probes, and culture media were from ThermoFisher (Cergy-Pontoise, France), and peroxidase-conjugated secondary antibodies were from Rockland Immunochemicals (Gilbertsville, PA). The control non-specific siRNA “ON-TARGETplus Non-targeting Control Pool”, MRCKα siRNA “ON-TARGETplus Human CDC42BPA siRNA SMARTpool”, Myl12a siRNA “ONTARGETplus Human Myl12a siRNA SMARTpool”, and Myl12b siRNA “ON-TARGETplus Human Myl12b siRNA SMARTpool” were from Dharmacon-GE Healthcare (Fontenay-sous-Bois, France). The ECL-Prime detection kit was from VWR (Courtaboeuf, France). The transfection reagents Turbofect and JetPrime were purchased from ThermoFisherScientific, (Saint-Herblain, France) and Ozyme (Saint-Cyr-l’Ecole, France), respectively. Chelerythrine chloride was obtained from Enzo Life Science (Villeurbanne, France).

Tissues were snap frozen in liquid nitrogen, crushed, and then lysed using RIPA Lysis Buffer System (Santa Cruz; sc-24948), according to the manufacturer’s instructions. Tissue lysates were cleared by centrifugation at 15 000g for 10 minutes at 4 °C. Protein concentration in the lysates was measured by BCA Protein Assay (Pierce; 23225) and normalized to the same concentration for each batch. Lysates were then diluted in nonreducing Laemmli sodium dodecyl sulfate sample buffer and heated at 95 °C for 5 minutes. Protein (30–50 μg) was loaded onto Mini-PROTEAN TGX Gels (Biorad; 4561096). After electrophoresis, protein was transferred onto polyvinylidene difluoride membrane using the BioRad Translotter system, and membranes were blocked for an hour in blocking buffer containing 5% bovine serum albumin. Membranes were then stained overnight at 4 °C with rabbit polyclonal anti-mouse FPN antibody (NBP1-21502; Novus Biologicals) at 1:1000 or horseradish peroxidase–conjugated anti–β-actin antibody (Proteintech; HRP-60008) at 1:5000. Blots were developed using the ECL prime detection kit (RPN2232; VWR International). Signal intensities were quantified by ImageJ, and the ratio between the FPN and the β-actin signals was calculated to produce normalized intensities.