FIG. 2.
Expression analysis of hRIPα and its isoforms and differential localization of hRIPα and hRIPβ. (A) In vitro translation of hRIPα and its isoforms. hRIPα and its isoforms were translated in vitro using [35S]methionine and analyzed by 15% SDS-PAGE. (B) Transient expression of hRIPα and its isoforms. Plasmids encoding hRIPα and its isoforms were transiently transfected into HEK293T cells. Equal amounts of protein were subjected to SDS-PAGE, and hRIPα and its isoforms were identified by immunoblotting (IB) with anti-HA antibody. Nontransfected cell lysate was also probed with anti-hRIP antibody and anti-hRIPAS antibody to show expression of the endogenous proteins. The star indicates nonspecific bands. (C) Differential localization of hRIPα and hRIPβ. HEK293T cells or COS cells were transiently transfected with plasmids encoding hRIPα or hRIPβ. The transfected cells were fixed and then immunostained with anti-hRIP or anti-HA antibody. DNA was visualized using propidium iodide staining. Bars, 10 μm. (D) Subcellular fractionation of hRIPα- and hRIPβ-transfected cells. Extracts from transfected cells were fractionated to give cytoplasmic (C) and nuclear (N) fractions as described in Materials and Methods. Aliquots of 5% of each fraction were immunoblotted with anti-HA (upper panel) and antilamin (bottom panel) antibodies. (E) hRIPβ localizes to the PML nuclear body. Cells were prepared as for panel A and immunostained with anti-hRIP antibody (green) and anti-PML antibody (red).