Mini protean stain free precast gels

Lysates and immunoprecipitates were analyzed by SDS-PAGE on mini-PROTEAN Stain-free Precast gels (Biorad, Marne la coquette, France), transferred to nitrocellulose membrane, and immunoblotted with primary antibodies, including anti-FLAG M2 (Sigma, Saint Quentin Falavier, France), anti-myc (9B11, Cell Signaling, Leiden, Holland), polyclonal anti-protein C (hpc4, Cell signaling), rabbit anti-GAPDH (Abcam, Cambridge, UK), polyclonal anti-GFP (gift from S. Miserey-Lenkei, Curie Institute; Paris, France), and polyclonal anti-KLHL21 (Invitrogen, Paris, France) antibodies. Thereafter, the membranes were incubated with a horseradish peroxidase-conjugated mouse or rabbit secondary antibody (1:5000 dilution). The images were obtained with chemiluminescence (Clarity Max Western EXL, Biorad; Marne la coquette, France) using a luminescent image analyzer (ChemiDoc XRS+, Biorad, Marne la coquette, France) and quantified with ImageLab software version 8.1.0 (Biorad, Marne la coquette, France).

Cells were treated with 40 μM of RSV with or without ALA for 24 h. For protein analysis, whole cell protein lysates from HepG2 were harvested with RIPA buffer (50 mmol/l Tris, 150 mmol/l NaCl, 0.5% sodium deoxycholate (v/v), 0.1% SDS (w/v), 1% NP-40 (v/v), pH 7.4) supplemented with proteinase inhibitors. Protein concentrations were determined with the Pierce™ BCA assay (Thermo Fisher Scientific GmbH, life technologies™, Darmstadt, Germany) according to the manufacturer's instructions and analyzed by Western Blotting analysis as described before (40 (link)). Briefly, 30 μg per samples were heated with loading buffer and applied to Mini-PROTEAN® Stainfree™ Precast Gels (4–20%, BioRad laboratories GmbH, Munich, Germany). Samples were transferred to a polyvinylidenedifluoride membrane (Bio-Rad laboratories GmbH, Munich, Germany) and blocked with skim milk dissolved in Tris-buffered saline + 0.05% (v/v) Tween 20. Membranes were then incubated with the primary antibodies FADS1, FADS2, CPT1, GAPDH (sc-134337, sc-98480, sc-48357, and sc-20357, respectively; Santa Cruz Biotechnology Inc., Dallas, USA) and FASN (bs-5045R, Bioss Inc., Massachusetts, USA). Secondary antibodies were from Santa Cruz Biotechnology Inc., Dallas, USA. Results were analyzed using Image Lab 5.0 (Bio-Rad laboratories GmbH, Munich, Germany).