FIG. 4.
9ORF1 complexes with MUPP1 in cells. (A) 9ORF1 complexes with HA epitope-tagged rat MUPP1 protein (HAMUPP1) expressed in COS7 cells. Cells were lipofected with 6 μg of GW1-HAMUPP1 plasmid and 2 μg of either empty GW1 plasmid (vector) or a GW1 plasmid expressing wild-type or the indicated mutant 9ORF1 protein. Cell proteins in RIPA buffer were either immunoblotted with HA antibodies or 9ORF1 antiserum (left) or first immunoprecipitated (IP) with 9ORF1 antiserum (α-9ORF1) and then immunoblotted with HA antibodies or 9ORF1 antiserum (right). Cell proteins at 100 or 800 μg were used in the experiment shown in the left and right panels, respectively. (B) 9ORF1 complexes with endogenous MUPP1 of CREF cells. Cell proteins in RIPA buffer were either immunoblotted with MUPP1 or 9ORF1 antiserum (top) or first immunoprecipitated (IP) with 9ORF1 antiserum or the matched preimmune serum (pre) (bottom left) or, alternatively, with either MUPP1 antiserum (α-MUPP1) or the matched preimmune serum (pre) (bottom right) and then immunoblotted with either MUPP1 or 9ORF1 antiserum. CREF cell proteins at 100 μg, 3 mg, and 3 mg were used in the experiments in the top, bottom left, and bottom right panels, respectively. (C) 9ORF1 complexes with MUPP1 in the Ad9-induced rat mammary tumor cell line 20-8. This tumor cell line contains a single integrated copy of the entire Ad9 genome (unpublished results). Cell proteins (3 mg) in RIPA buffer were first immunoprecipitated (IP) with either 9BP-1 antiserum (α-9BP-1) or the matched preimmune serum and then immunoblotted with either 9ORF1 or 9BP-1 antiserum.