1. Lysates ( 293 cells transfected with Mit Bcl3 hi or Mit alone reduced with 20mM bME) were loaded at 10^6 cell equivalents per lane gel and separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane using Idea Scientific electrophoresis system according to the manufacturer'??s instructions.
2. Nitrocellulose blots were blocked with a solution of 5% nonfat milk in Tris-buffered saline for 1h at room temperature.
3. The blots were incubated at 4C for 18h with primary antibody (NB 100-352) at 1 ug/ml.
4. The blots were incubated with HRP-linked secondary antibody for 1h at RT.
5. Immunoreactive bands were visualized using the enhanced chemiluminescence light (ECL) detection reagents (Amersham, Arlington Heights, IL).
Note: The membranes were washed before and after incubating with the primary antibody and before developing the film in TBS-T (Tris buffered saline with Tween-20).