Fig. 3. SidI interacts with components of the mammalian translation elongation machinery.

A. Components of the host protein synthesis machinery were retained by SidI. Affigel beads coated with GST (lane 1) or His₆-SidI (lane 3) were incubated with mammalian cell lysates. His₆-SidI coated beads incubated with cell lysis buffer (lane 2) was used as another control. After washing with lysis buffer, proteins separated by SDS-PAGE were visualized by silver staining; bands only retained by the His₆-SidI coated beads were identified by MALDI /mass spectrometry analysis. Relevant protein size markers (in kDa) were indicated. B. Co-purification of eEF1A and eEF1Bγ with GST-SidI from rabbit reticulocyte lysates (RRL). Each GST-tagged protein was incubated with RRL and glutathione coated beads were used to retrieve GST-SidI or GST-SidF. After removing unbound proteins by extensive washing, the retained proteins resolved by SDS-PAGE were detected by Coomassie bright blue staining and identified by mass/spectrometry analysis. Relevant markers (in kDa) were indicated. C. Direct binding of SidI to eEF1A and eEF1Bγ. His₆-SidI was incubated with GST-tagged eEF1A, eEF1Bγ or eEF1Bβ, and the protein complex was captured with glutathione beads, retained SidI was detected by immunoblotting. D. SidI* formed complexes with eEF1A or eEF1Bγ in mammalian cells. Lysates of 293T cells transfected to express GFP-SidI* or GFP-SidF were subjected to immunoprecipitation with an anti-GFP antibody, the precipitates resolved by SDS-PAGE were detected for eEF1A and eEF1Bγ using specific antibodies. 5% (50 μg) of total protein was probed as input controls (lanes 1 and 2). TCL: total cell lysates.