Fig. 3.
The LCCD region interacts with SSDP1 and RLIM and mediates instability. (A) The LCCD domain functions as an instability domain. mRNA encoding Myc-LCCD was injected in zebrafish embryos. Embryos were fixed at 5 and 24 hpf and stained with a monoclonal Myc antibody. Note that little or no Myc staining is detected in 24 hpf embryos of Myc-LCCD-injected animals, whereas Myc-NLS control proteins are readily detectable. (B) The LCCD region interacts directly with RLIM in vitro. Shown is GST pull-down using GST-RLIM and [35S]Myc-LCCD. (C) The LCCD interacts with RLIM and SSDP1 in cells. Co-IP of SSDP1 and RLIM from αT3 cells transfected with Myc-LCCD. (Upper) Western blot of Myc IPs (Left) and 100% input control (Right) using antibodies directed against RLIM and SSDP1 (10% gel). (Lower) To visualize Myc-proteins, an aliquot of the same Myc-IPs was run on a 15% gel in parallel. Myc proteins are indicated by asterisks. Lower Myc-LCCD and Myc-CLIMΔC protein levels probably reflect their instability in cells. Bands not marked with asterisks are unspecific.