Immunoblotting Analysis of KLF5 in B-cell Precursors

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).