Mouse monoclonal anti lamin a c antibody

Cells were treated with 200 μM H₂O₂ for 0 to 12 h. After washing with HBSS, cells were homogenized in cell lysis buffer to obtain the nuclear fraction using Nuclear Extraction Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. The following separation, blotting, detection and visualization procedures were performed in the same manner as for MAPK phosphorylation immunoblotting. Target proteins were detected with mouse monoclonal anti-IL-33 antibody (1:1000 dilution, Enzo Life Sciences, Exeter, UK) or mouse monoclonal anti-lamin A/C antibody (1:400 dilution; Santa Cruz Biotechnology, Santa Cruz, CA).

Cells were seeded in 60 mm dishes at a density of 1 × 10⁵ /ml. At 90 % confluence, the cells were treated with various concentrations of H₂O₂ (Wako, Osaka, Japan) or ONOO⁻ (Calbiochem, La Jolla, CA). To obtain the nuclear fractions, a Nuclear Extraction Kit (Active Motif, Carlsbad, CA) was used according to the manufacturer’s instructions. The cells were washed with ice-cold PBS and homogenized in cell lysis buffer. Equal amounts of protein were loaded and separated by electrophoresis on 12 % SDS polyacrylamide gels. The separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). The following antibodies were used for detection of the target proteins: rabbit polyclonal anti-KLF5 antibody (1:1000 dilution, Abcam), mouse monoclonal anti-β-actin antibody (1:4000 dilution, Sigma-Aldrich, St. Louis, MO), mouse monoclonal anti-lamin A/C antibody (1:400 dilution, Santa Cruz Biotechnology, Dallas, TX), mouse monoclonal anti-α-SMA (1:5000 dilution, Sigma-Aldrich). Bound antibodies were visualized using the appropriate peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (GE Healthcare Life Sciences, Buckinghamshire, UK) with a chemiluminescene imaging system (LAS-4000 mini, Fujifilm, Tokyo, Japan). Band intensity was quantified by densitometry (Quantity One, Bio-Rad Laboratories).