Anti β actin mouse monoclonal antibody

SH-SY5Y cells and mouse brain total protein were prepared by homogenization in RIPA (Radioimmunoprecipitation Assay) Buffer (Sigma) with 1X protease inhibitor cocktail (Roche, Nutley, NJ). The nuclear and cytosolic fractions from LPS- or LPS plus celastrol-treated SH-SY5Y cells were obtained using the NE-PER nuclear and cytoplasmic extraction kit (ThermoFisher Scientific). Total protein (60 μg) was separated using 4-12% NuPAGE Bis-Tris protein gels (Invitrogen) and transferred to a PVDF membrane. After protein transfer, the membrane was blocked for 10 min at room temperature in LI-COR Odyssey Blocking Buffer and then probed with previously characterized primary SVCT2 antibodies (1 : 500 dilution) [40 (link)], anti-IKKαβ antibodies (1 : 1000 dilution; Abcam), anti-NF-κβ p65 antibodies (1 : 1000 dilution; Abcam), anti-laminin antibodies (1 : 300 dilution; Santa Cruz Biotechnology), and anti-β-actin mouse monoclonal antibody (1 : 3000 dilution; ThermoFisher Scientific) used. The respective secondary antibodies (anti-rabbit IRDye-800 and anti-mouse IRDye-680, LI-COR Biosciences) were used in 1 : 30,000 dilutions [33 (link), 34 (link), 40 (link)]. Odyssey Infrared imaging system (LI-COR Biosciences) software was used to quantify the densitometry of specific band signal intensities normalized against β-actin.

Caco-2 cells were lysed in lysis buffer (CelLytic Mammalian Cells, Sigma Aldrich^®, Saint-Quentin Fallavier, France) containing protease inhibitors (Complete, Roche^®, Meylan, France). The protein concentration was measured using the BCA colorimetric method (BC Assay Protein Quantitation Small Kit, Interchim^®, Montluçon, France) and, for each experimental condition, an equal amount of total proteins was separated on 8% SDS polyacrilamide gel and then transferred onto PVDF membranes. Blots were blocked overnight in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skimmed milk (Sigma Aldrich^®, Saint-Quentin Fallavier, France). The PVDF membranes were further stained for 1 h at room temperature with a mouse monoclonal anti β-actin antibody (Ambion, Life technology^®, Saint Aubin, France) or a mouse monoclonal anti-ZO-1 antibody (Invitrogen, Life Technology^®, Saint Aubin, France). Blots were then incubated for 1h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Dako, Agilent Technology^®, Les Ulis, France) and revealed by chemiluminescence (ECL plus, Amersham, GE Healthcare Life Science^®, Velizy-Villacoublay, France) before printing on radiographic film (Hyperfilm, Amersham, GE Healthcare Life Science^®, Velizy-Villacoublay, France).