Anti β actin antibody

MC3T3-E1 was treated with ALA and SFC for 1 h and then treated with LPS for 24 h. Brefeldin (3.0 μg/ml) were used to inhibit protein transport during culture. Cells were washed with PBS and treated with cell lysis buffer (5 mM EDTA, 10％ glycerol, 1％ Triton X-100, 0.1% SDS, 1％ NP-40) in PBS. Protein concentration in each of the lysates was measured with Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA) and adjusted to be the same for each lysate. After mixing with sample buffer, it was heat denatured, were electrophoresed on a TGX Precast gel (Bio-Rad Laboratories). The proteins were transferred to a PVDF membrane, and blocked with PVDF Blocking Reagent (Toyobo Co. Ltd, Osaka, Japan). Membrane was then incubated with anti-IL6 antibody (1/2000 dilution; ProteinTech Group, Chicago, IL, USA). After washing 0.5% Tween-20 in PBS (PBS-T), the membrane was incubated with HRP-conjugated secondary antibody (Thermo Fishter Scientific, San Jose, CA). To confirm the amount of the loaded protein were equal, membrane was incubated with anti-β Actin antibody (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Chemiluminescence was produced using Luminata Forte (EMD Millipore Corporation, Billerica, MA) and detected with LumiCube (Liponics, Tokyo, Japan).

Cell lysates were prepared using NP-40 lysis buffer (0.5% Non-idet P-40, 5 mM EDTA, 2 mM Na₃VO₄, 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mg/mL aprotinin, 1 mM PMSF). Protein concentration was determined using the Protein Assay Dye (Bio-Rad Laboratories, Hercules, CA, United States). Proteins (20 μg) were separated using 12% SDS-PAGE and transferred to a PVDF membrane (GE Healthcare). The membrane was probed with anti-β-catenin antibody (#8480; Cell Signaling, Danvers, MA, United States) or anti-β-actin antibody (013-24553; Fujifilm Wako Pure Chemicals, Osaka, Japan). Horseradish peroxidase-labeled anti-rabbit IgG antibody (GE Healthcare) and anti-mouse IgG antibody (GE Healthcare) were used as the secondary antibodies. The proteins were detected using an ImmunoStar LD chemiluminescence detection kit (Fujifilm Wako Pure Chemicals) and visualized with a LAS-1000 Lumino Image analyzer (Fujifilm, Tokyo, Japan).