Anti klf5 antibody

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).

FACS sorted purified B-cell precursors or ALL cell line cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and extracts were electrophoresed on SDS-PAGE. For immunoblotting, separated proteins were transferred to a PVDF membrane. The membrane was then blocked with 5% non-fat milk in tris-buffered saline (TBS) for one hour at room temperature (RT). A guinea pig anti-KLF5 antibody [31 (link)] (1:4000) was used for mouse samples, anti-KLF5 antibody (Abcam, Cambridge, MA, Catalog # ab24331,1:1000) for human ALL cell line samples, c-Abl antibody (Cell Signaling Technology, Catalog # 2862, 1:1000), Gstm1 antibody (Santa Cruz Biotechnology, Catalog # sc-133641, 1:500) was added separately and incubated overnight at 4°C. Mouse anti-β-actin antibody (Sigma) was added as a loading control. The filters were washed, incubated with a secondary anti-mouse, rabbit or guinea pig HRP-conjugated antibody for one hour at room temperature and the bands were visualized using enhanced chemiluminescence (Amersham ECL, GE Healthcare).