Figure 2.
Effects of MSG1 on transcriptional activation mediated by Smad4 C-terminal domain (amino acids 302–552) in NIH 3T3 cells. (A) Cells were cotransfected with expression plasmids for GAL4 DNA-binding domain (DB) fusion proteins and activators together with a GAL4-dependent CAT reporter plasmid (pG5CAT), followed by incubation in the presence of 10% fetal calf serum for 16 h and determination of relative CAT activity (Rel.CAT). (B) Cells were cotransfected with GAL4DB-Smad4[302–552], HA epitope-tagged MSG1 and pG5CAT, followed by incubation in serum-free medium supplemented with TGFβ1 for 16 h and CAT assay. (C) Cells were cotransfected with GAL4DB-Smad4[302–552] and pG5CAT together with HA-MSG1, HA-TβRI(T204D) activated TGFβ type I receptor kinase, Flag epitope-tagged Smad2, or Flag-Smad3, followed by incubation in serum-free medium for 16 h and CAT assay. Protein expression of MSG1 and TβRI(T204D) (B and C) was evaluated by anti-HA Western blotting.