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. 2007 Mar 14;104(12):5121–5126. doi: 10.1073/pnas.0611030104

Fig. 4.

Fig. 4.

SidF interacts with BNIP3 and Bcl-rambo. (A) Interactions between SidF and Bcl2-rambo/BNIP3 in yeast two-hybrid assay. Yeast strains harboring the indicated constructs were streaked on Leu− and Trp− synthetic medium to select for plasmids (on the left) or on Leu−, Trp−, Ade−, and His− medium to examine the interactions (on the right). The two SidF deletion mutants tested were SidFΔN1, SidF250–912 and SidFΔN2, SidF650–912. (B) SidF forms complexes with BNIP3. Cell lysates of 293T cells transfected with the indicated plasmid combinations were analyzed either directly or after immunoprecipitation by immunoblots with an anti-FLAG antibody. Relevant molecular mass markers are shown on the left in kilodaltons. (C) SidF in total cell lysates was detected by anti-SidF antibody. (D–F) SidF interacts with Bcl-rambo in mammalian cells. (D) 293T cells were transfected with the indicated plasmid combinations, and cell lysates were used for immunoprecipitation. Flag-Bcl-rambo was detected with an anti-FLAG antibody. (E) SidF forms a complex with endogenous Bcl-rambo. SidF was probed in immunoprecipitates obtained with anti-Bcl-rambo antibody from cells transfected with shown plasmid combinations. Note that more SidF was detected in precipitates of cells transfected with a Bcl-rambo-expressing plasmid. (F) SidF and Bcl-rambo form a complex in cells infected by a Dot/Icm-competent strain overexpressing SidF. Lysates of U937 cells infected with wild-type (lane 1), dotA−(pSidF) (lane 2), the sidF− mutant (lane 3), or ΔsidF(pSidF) (lane 4) for 6 h were subjected to immunoprecipitation with anti-Bcl-rambo antibody, and the presence of SidF/Bcl-rambo complexes was probed with anti-SidF antibody. (G and H) SidF directly binds Bcl-rambo. (G) Purification of GST-Bcl-rambo. Relevant molecular mass markers are shown on the left in kDa. (H) Binding between Bcl-rambo and SidF. Glutathione beads or beads coated with GST or GST-Bcl-rambo were incubated with SidF for 4 h at 4°C. After extensive washing, bound proteins were eluted with SDS loading buffer, resolved by SDS/PAGE, and probed with anti-SidF antibody (Lower). Ten percent of input proteins were detected by Coomassie bright blue staining (Upper).