Figure 4.

The effect of H₂O₂ treatment of Raji cells on BSAP/Pax5 DNA-binding activity. (A) Induction of BSAP/Pax5 DNA-binding activity by treatment of B cells with H₂O₂. Raji cells were left untreated (lane 2) or incubated for 30 min with 50 µM H₂O₂ (lane 3). Nuclear extracts (lanes 2–3) were prepared as described in Materials and Methods. An aliquot of 5 µg of protein was reacted with a ³²P-labeled H2A-2.2 probe (25). Samples were analyzed on a native 7% polyacrylamide gel. An autoradiograph of the gel is shown. The arrow indicates the position of the BSAP/Pax-5–DNA specific complex. To test for the specificity of the complex, a competition assay with cold specific and non-specific oligonucleotide was performed. As expected, the specific H2A-2.2 oligonucleotide efficiently reduces the BSAP/Pax-5–DNA complex (lanes 4 and 5), whereas the non-specific BS2 oligonucleotide is almost ineffective (lanes 6 and 7). (B) The expression levels of BSAP/Pax-5 are not induced by a 30 min treatment of B cells with H₂O₂. The nuclear samples, as in (A) (10 µg/lane), were loaded onto a SDS–PAGE gel for western blot analysis with anti BSAP/Pax-5 monoclonal antibody. The blot was developed using an ECL chemiluminescence detection kit (Amersham Biotech).