FIG. 5.

Chronological expression profile of RTA, responsiveness of monomer RE I-2B and RE IIC-2, and the binding activity of α50A in EMSA. (A) NE was prepared from BCBL1 cells at 0, 2, 4, 8, 12, 24, 48, and 72 h postinduction with TPA. A total of 20 μg of the NE was subjected to Western blotting analyses. RTA was detected with a mouse monoclonal antibody to RTA (α50A) as the first antibody and an anti-mouse IgG Fab fragment conjugated with horseradish peroxidase (see Materials and Methods). (B) Monomer constructs of RE I-2B and RE IIC-2 were transfected into 293L cells with β-galactosidase expression vector (pCMVβ) for normalization of transfection efficiency. The fold activity was calculated as mentioned above (see Materials and Methods). (C) EMSA was performed with PAN RRE as a probe. Cold competitors such as PAN RRE, RE I-2B, and RE IIC-2 were added to the reaction mixture in 50-fold excess for each case. Next, 2 μg of specific antibody to RTA (α50A) and Oct1 (αOct1) was added for supershift and/or binding inhibition analysis. The arrow denotes a supershifted complex with α50A. PI, preimmune serum.