FIGURE 1.

Human CTIF interacts with eIF3g. A, yeast two-hybrid analysis of human CTIFΔN and eIF3 subunits. Yeast strain (PBN204) was co-transformed with plasmids expressing BD-CTIFΔN and AD-eIF3 subunits (a–k). Transformed yeast cells were spread on selective medium lacking leucine and tryptophan (SD-LW) to select for co-transformants (Master plate). Specific interactions between two proteins were tested as follows: (i) by the appearance of visible blue color by LacZ expression (Filter assay); (ii) growth on selective medium lacking leucine, tryptophan, and uracil (SD-LWU), and (iii) growth on selective medium lacking leucine, tryptophan, and adenine (SD-LWA). The dimerization of polypyrimidine tract-binding protein served as the positive control (+). The empty vector pBCT and pACT2 served as the negative control (−). B, in vitro GST pulldown assays of CTIF(365–598). Extracts of E. coli expressing GST, GST-eIF3g, and GST-AMSH were mixed with purified recombinant His-CTIF(365–598). After pulldown using GST resin, the precipitated proteins were analyzed by Western blotting (WB) using α-GST antibody (upper panel) or α-His₆ antibody (lower panel). The locations of markers for molecular weight are indicated on the left. C, far Western blotting analysis (FW) of purified eIF3 complex. Left panel, Coomassie Blue staining results showing the integrities and relative abundances of input proteins. The degraded product of eIF3a is denoted as ΔeIF3a. Right panel, purified rabbit eIF3 complex was resolved by SDS-PAGE. The purified His-CTIF(365–598) and α-His₆ antibody were used as a probe and a primary antibody, respectively. D, far Western blotting analysis using recombinant GST-eIF3g. As in C, except that BSA, recombinant GST-AMSH, and GST-eIF3g were resolved by SDS-PAGE.