Modulation of CP2 Family Transcriptional Activity by CRTR-1 and Sumoylation
Figure 2
Mapping of CRTR-1 transactivation and repression domains.
A. Schematic diagram of the CRTR-1 truncations fused to the GAL4 DNA-binding domain. Numbers in brackets represent the first and last amino acid of the CRTR-1 protein included in the protein. B. Luciferase reporter assays of GAL4-CRTR-1 deletion constructs. HEK293T cells were co-transfected with 200 ng pTK-MH100x4-LUC and 200 ng of expression plasmid for the GAL4-CRTR-1 deletion mutants together with 5 ng of pRL-SV40 for normalizing transfection efficiency. The data are presented relative to the activity of the reporter construct alone and are the mean ± SEM of three independent experiments, each of which was conducted in triplicate. Statistical significance was determined using a two-tailed unpaired t-test comparing the activities of each GAL4-CRTR-1 deletion mutant with GAL4 vector alone. ** denotes statistical significance with P<0.01; *** denotes P<0.0001. C. Expression level of GAL4-CRTR-1 deletion mutants. Whole cell lysates from HEK293T cells transfected with 2 µg of expression plasmid encoding GAL4-CRTR-1 deletion mutants were immunoblotted with an anti-GAL4 antibody and detected by ECF (upper panel). The membrane was re-probed with rat anti-alpha-tubulin antibody and detected by enhanced chemiluminescence (lower panel). The predicted sizes of the GAL4-CRTR-1 fusion proteins are as follows: GAL4-CRTR-1(1–47), 21 kD; GAL4-CRTR-1(48–479), 64 kD; GAL4-CRTR-1(101–479), 58 kD; GAL4-CRTR-1(198–479), 47 kD; GAL4-CRTR-1(48–200), 33 kD; GAL4-CRTR-1(101–200), 27 kD; GAL4-CRTR-1(1–100), 27 kD; GAL4-CRTR-1(1–200), 38 kD; GAL4-CRTR-1(1–479), 69 kD; and GAL4-CRTR-1(1–52), 22 kD. Specific bands corresponding to GAL4-CRTR-1 deletion mutants are marked with an asterisk. Mass of molecular weight markers (kD) are shown.