Cellular location and homodimerization analyses of STAT3-RARA (SR) fusion protein. (A) Immunofluorescence analysis of 293T cells transfected with pcDNA3.1 expression plasmids of vehicle (vector), hemagglutinin (HA)-tagged STAT3, HA-tagged RARA, HA-tagged SR, and MYC-PML-RARA, respectively. HA antibody was used as primary antibody, and 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. (B) ATRA downregulates SR protein expression. After transfection with pcDNA3.1 HA-tagged SR, 293T cells were treated with 1 μM of ATRA or dimethyl sulfoxide (DMSO; vehicle control) for 48 hours. The tagged protein was detected using an anti-HA antibody followed by an Alexa Fluor 488–conjugated secondary antibody. DAPI was used for nuclear staining. (C) Viral-transfected 293T cells were treated with 1 μM of ATRA or DMSO (vehicle control) for 48 hours before western blot. The tagged protein was detected using an antiflag antibody, and β-actin served as the loading control. (D) Empty-vector, flag-tagged SR T1 and T2 were expressed in 293T cells. Immunoblotting analysis of expression of flag-tagged fusions, STAT3, phosphorylated (P) STAT3, AKT, P-AKT, MAPK, P-MAPK, BCL2, c-MYC, cyclin D1, and caspase 3 was performed in 293T cell. Protein level of STAT3 targets with SR expression. (E-F) Homodimerization of SR fusions were detected by coimmunoprecipitation. Both HA-tagged SR T1 (E) and HA-tagged SR T2 (F) can be immunoprecipitated by flag antibody and vice versa. IP, immunoprecipitation.