Anti laminin antibodies

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.

The ¹⁴C-AA (specific activity 2.8 mCi/mmol; radiochemical purity > 98%) used in vitamin uptake analysis was acquired from PerkinElmer, Inc. (Boston, MA). LPS (E. coli 0111:B4) was purchased from Millipore Sigma (St. Louis, MO). Human TNFα was bought from Invitrogen (Carlsbad, CA), and murine TNFα was from PeproTech, Inc. (Rocky Hill, NJ). Antibodies were obtained from the following sources: anti-β-actin antibodies (ThermoFisher Scientific, Huntington Beach, CA), anti-NF-κB p65 and anti-IKKαβ antibodies (Abcam, Cambridge, MA), and anti-laminin antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). LI-COR (Lincoln, NE) IRDyes 800CW and 680LT goat anti-mouse and anti-rabbit secondary antibodies were used for western blot. Celastrol was ordered from InvivoGen, Inc. (San Diego, CA). Services provided by Integrated DNA Technologies (San Diego, CA) were used to synthesize oligonucleotide primers (Table 1). All other molecular biology-grade chemicals, reagents, and materials used in this study were from commercial sources.