C abl antibody

K562 and KA were treated with different concentrations of DMP or at different time points. After adding the lysate (50 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.1 mM sodium orthovanadate, 50 μg/mL aprotinin, 1 mM phenylmethysulfonyl fluoride, and 10 mM Tris-HCl; pH 7.4), shock lysis was performed on the ice bath for 30 min, followed by ultrasonic nucleation on the ice bath for 30 seconds (50% strength, 2 s/4 s), centrifugation at 12000 rpm at 4°C for 15 min. After the supernatant was taken, the protein was quantified and SDS-PAGE gel electrophoresis was performed. After electrophoresis, the samples were transferred to nitrocellulocellulose membrane, followed by an immune reaction, and then incubated with c-Abl antibody (Cell Signaling Technology #2862), SRC antibody (Cell Signaling Technology #2108), β-actin (Cell Signaling Technology #8457) at room temperature for 4°C overnight. Goat anti-rabbit IgG-HRP (Cell Signaling Technology) was incubated for 2 hours, TBST was washed for 2 hours, and ECL solution was added for 1 minute. The membrane was drained and exposed in a bio-RAD chemiluminescence imager for several minutes. The results were read by ImageLab 5.2.1 software and analyzed statistically with β-actin as the internal reference.

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).

NIT-1 cells were spun to the slides by cytospinning. The cells in the slides were fixed with 4% paraformaldehyde for 5 minutes and then blocked for 10 minutes at room temperature with 10% normal goat serum. The cells were spun onto the glides and stained with c-abl antibody for 1 hour at room temperature (1∶200, Cell Signaling Technology) followed by secondary antibody, goat anti rabbit AF555 for 1 hour at room temperature (1∶1000, Invitrogen Technology). All the washing steps were used with 1X TBS solution. Imaging was done using Zeiss Axioskop microscope.