APH-2 binds to CREB but not to p300 and is expressed in vivo. (A) Cell lysates (400 μg) obtained from 293T cells transfected with pcDNA3.1/Myc backbone vector (lane 1), APH-2-Myc (lane 2), or HBZ-SP1-Myc (lane 3) were subjected to immunoprecipitation with a rabbit polyclonal anti-CREB antibody followed by Western blot with a mouse monoclonal anti-Myc antibody. Input proteins (50 μg) are shown in the middle (anti-Myc) and bottom (anti-CREB) panels. (B) Purified p300 (0.2 pmol) was incubated with 5 pmol GST, GST-HBZ, or GST-APH-2. Bound proteins were detected by Western blot analysis with an anti-p300 antibody. Input protein (100%) is shown in lane 1. (Bottom panel) Purified GST, GST-HBZ, and GST-APH-2 (5 pmol of each) are shown by Coomassie blue staining after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Lane 1 represents molecular weight marker. (C) Cell lysates (400 μg) obtained from MO (lane 1), Jurkat (lane 2), and C91PL (lane 3) were subjected to immunoprecipitation with a rabbit polyclonal anti-CREB antibody followed by Western blot with a rabbit polyclonal anti-APH-2 antibody. Input proteins (50 μg) are shown in the bottom (anti-CREB) panels. (Top panel) These lanes correspond to different part of the same gel.