Figure 4

ORF2 protein associates with βTRCP protein. (A) Huh7 cells were transfected with pSGI, ORF2 and myc tagged βTRCP expression constructs in indicated combinations and labeled with [³⁵S]-cys/met promix. Lysates were immunoprecipitated with anti-ORF2 (lane 1 and 2) or anti-Myc (lane 3) antibody and immunoblotted with anti-Myc antibodies. Immunoblot presented in upper panel was air dried and exposed to X-ray film to check for ORF2 expression (lower panel). (B) Huh7 cells transfected with pSGI vector or ORF2 or c-Myc expression constructs were labeled with [³⁵S]-cys/met promix, immunoprecipitated with the indicated antibodies and immunoblotted with anti-SKP2 or anti-Myc antibodies. Myc blot was air dried and exposed to X-ray film to check for ORF2 expression (3^rd panel). (C) Lysates from mock (lane 2), ORF2 (lane 3 and 6), Δ35 ORF2 (lane 4) and KDEL ORF2 (lane 5) transfected Huh7 cells, were immunoprecipitated using anti-ORF2 antibody (lane 2–5) or pre-immune serum (PS, lane 6) and incubated along with in vitro translated [³⁵S]-cys/met labeled full-length βTRCP protein (lane 2–6). Bound βTRCP protein band was visualized by autoradiography. Lane 1 represents total βTRCP protein used in each sample. For negative control, ORF3 expressing cells were immunoprecipitated with anti-ORF3 antibody and processed as above (lane 7).