Fusion capt software

HepaRG cells were treated with FLX alone or co-treated with the different inhibitors. After washing with cold PBS, they were suspended in cell lysis buffer supplemented with protease and phosphatase inhibitors (Roche, Mannheim, Germany). Aliquots containing an equivalent total protein content as determined by the Bradford procedure with bovine serum albumin as the standard were subjected to sodium dodecyl sulfate/12% polyacrylamide gel electrophoresis, electrotransferred to Immobilon-P membranes, and incubated overnight with primary antibodies directed against p-MYPT1 (dilution: 1/500), p-AKT (dilution: 1/2000), total AKT (dilution: 1/2000), p-P38 (dilution: 1/1000), total P38 (dilution: 1/1000), p-HSP27 (dilution: 1/500), total HSP27 (dilution: 1/1000) or HSC70 (dilution: 1/500). After exposure to a horseradish peroxidase conjugated anti-mouse/rabbit antibody (dilution: 1/5000) (Thermo Fisher Scientific, Waltham, MA), the membranes were incubated with a chemiluminescence reagent (Millipore, Billerica, MA) and the bands were then visualized with Fusion-Capt software (Vilber Lourmat, Collégien, France) and quantified using ImageJ 1.48 software.

For western blot detection of P53, P16, and P21, proteins were solubilized in 8 M Urea lysis buffer (8 M Urea, 50 mM Tris-HCl, 0.1 M β-Mercaptoethanol, 1 mM DTT, 100 μg/ml phenylmethanesulfonylfluoride). Proteins (15 μg) were separated by 8% (P53) or 14% (P21, P16) SDS-PAGE, blotted onto polyvinyl difluoride (PVDF) membrane (Millipore, Corporation, Billerica, MA, USA), and revealed using the anti-P53 DO-7 mAb (1/1000; Dako, Glustrup, Denmark), the anti-P21 EA10 mAb (0.5 μg/ml; Abcam ab16767, Cambridge, UK), or the anti-human-P16 mAb (1/1 000, BD Biosciences, Le Pont de Claix, France). Membranes were re probed using the anti-GAPDH mAb (1/2000; Abcam 9484, Cambridge, UK) or the anti-β-Tubulin TUB2.1 mAb (1/5 000, Sigma Aldrich, St Louis, MO) for loading/transfer controls. Membranes were then analysed using the Luminata crescendo western HRP substrate (Millipore Corporation, Billerica, MA, USA). Quantification of P53 was performed using the ImageJ software. Quantification of P21 and P16 was performed using the Fusion-Capt software (Vilber Lourmat, Eberhardzell, Germany). For detection of SHH, 1μg of concentrated culture supernatants were subjected to 10% SDS-PAGE, blotted, and revealed using the N-SHH 167Ab polyclonal antibody [26 (link)]. Quantification of N-SHH was performed using GeneGnome device and GeneTools software (Syngene, Synoptics Ltd, Cambridge, UK).

Prostate cancer cells were lysed in a buffer containing 10 mM Tris/HCl pH 7.6, 5 mM EDTA, 50 mM NaCl, 50 mM NaF and 1% Triton X100 (v/v). Cell nuclei and debris were removed by centrifugation (3 min, 10,000 x g), and the supernatant was tested for protein concentration using the BCA-method (Sigma Aldrich, Taufkirchen, Germany). Cell extracts were electrophoresed through a 10% SDS-PAGE gel (Novex, Carlsbad, CA, USA) and electroblotted onto a nitrocellulose or PVDF (Pall, Bad Kreuznach, Germany and Merck Millipore, Darmstadt, Germany). AR protein was detected by monoclonal mouse antibody AR441 (Dako, Hamburg, Germany, 1∶2000), detection of β-actin with mouse monoclonal Anti-β-Actin (Sigma Aldrich, Taufkirchen, Germany, 1∶2000) served as loading control. Immunoreactive bands were localized using horseradish peroxidase-labeld goat anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1∶2000-1∶10000). Immunoreactive complexes were visualized by direct detection of chemoluminescene using the Fusion FX7 Imaging and Gel documentation system and FUSION-CAPT software (Vilber Lourmat, Marne-la-Vallee, France).

Tissue lysate was harvested in a lysis buffer (25 mM bicine, 150 mM sodium chloride, pH 7.6; Pierce), homogenized, and centrifuged for 20 min at 12 000 rpm at 4°C. Nuclear and cytoplasmic fractions were prepared from renal tissue using a Nuclear Protein Isolation-Translocation Assay Kit (FIVEphoton Biochemicals, San Diego, CA, USA) according to manufacturer instructions. The protein concentration was detected using a BCA protein assay kit (Pierce BCA assay, Thermo Scientific, Rockford, IL). Proteins (30 or 50 μg) were separated using 8% –12% SDS-PAGE and then transferred to PVDF membranes (Pall Corporation). Blots were then probed with monoclonal anti-CAV-1 (1:1000; Epitomics), monoclonal Cyp A (1:1000; GeneTex), monoclonal anti-PGC-1α (1 μg/mL; Abcam), polyclonal anti-NRF-1 (1:1000; GeneTex), polyclonal anti-SOD2 (1:1000, Novus), polyclonal anti-catalase (1:1000; Abcam), MitoProfile Total OXPHOS rodent antibody cocktail (1:800; MitoSciences, Eugene, OR), monoclonal anti-Nrf2 (1:800; GeneTex), polyclonal anti-Keap1 (1:1000; Bioss), polyclonal anti-GCLC (1:800; GeneTex), polyclonal anti-Lamin B1 (1:1000; Abcam), mouse anti-GAPDH (1:1000; Abcam), and mouse anti-β-actin (1:10000; Millipore, Billerica, MA, USA). Signals were obtained using an enhanced chemiluminescence kit (Millipore), and densitometry was performed using Fusion-Capt software (Vilber Lourmat, Fusion FX7, France).

Total proteins were extracted from primary mammary epithelial cells of Dlc1^gt/+ and WT mice and 70 μg of lysates were separated by SDS-PAGE. The proteins were then transferred to Immobilon-P PVDF (polyvinylidene difluoride) membrane (Millipore, ON] for determination of specific protein expression levels using anti-rabbit Dlc1 (Santa Cruz Biotechnology, Dallas, TX,1:1000, cat#sc-32931,] using chemiluminescence as described by [32 (link)]. The blots were visualized by incubation with SuperSignal West Femto Substrate (Thermo Scientific, Rockford, IL) in the Fusion FX Gel Documentation system (Vilber Lourmat, Germany). The signal intensities were determined using the Fusion-CAPT software (Vilber Lourmat, Montreal Biotech, Dorval, PQ), and Dlc1 protein expression levels were determined as a ratio to β-actin (1:10,000 dilution, Sigma, St. Louis, MO).

Caecal tissue was disrupted and homogenized in a buffer containing 186 mM β-mercaptoethanol, 1% bromophenol blue, 10 mM NaF, 25 mM NaPPi, 1 mM Na3VO₄, using a microtube adapted pellet mixer. After lysis, samples were incubated at 100 °C for 10 min and centrifuged to remove insoluble materials. Proteins were resolved by SDS–PAGE, and gels were transferred to polyvinylidene difluoride (PVDF) membrane (GE Healthcare). For immunoblotting, membranes were washed with TBS 0.1% Tween 20, blocked in TBS (0.1% Tween 20, 5% milk) and probed overnight with the following specific antibodies: anti-ERK1/2, anti-JNK, anti-IκBα, anti-phosphorylated (anti-p)ERK1/2 and anti-pJNK (Cell Signaling Technology), or anti-actin (Millipore). Blots were then incubated with HRP-linked secondary antibodies (Cell Signaling Technology), followed by chemiluminescence detection with the ECL Plus kit (Millipore) according to the manufacturer’s instructions. Chemiluminescence signals were detected with a Fusion FX (Vilber Lourmat) and analyzed densitometrically with Fusion-CAPT software (Vilber Lourmat).