Figure 1. Identification of LZTFL1 as a binding partner of intraflagellar transport protein 27 (IFT27), a major spermatogenesis regulator.

A. Direct yeast two-hybrid assay to examine the interaction between IFT27 and LZTFL1. Pairs of indicated plasmids were co-transformed into AH109 yeast, and the transformed yeast were grown on either selection plates (lacking leucine, histidine and tryptophan) or non-selection plates (lacking leucine and tryptophan). Notice that all the yeast except AH109 grew on the non-selection plate. Yeast expressing IFT27/LZTFL1 and P53/large T antigen pairs grew on selection plate. B. LZTFL1/MYC overlaps with IFT27/GFP in CHO cells. When expressed alone, IFT27/GFP was present in the whole cells (a), and LZTFL1/MYC was present in the cytoplasm (b). When the two proteins were co-expressed, LZTFL1/MYC partially overlapped with IFT27/GFP in the cytoplasm (c). C. Co-immunoprecipitation of LZTFL1/MYC with IFT27/GFP. COS-1 cells were transfected with plasmids to co-express LZTFL1/MYC and IFT27/GFP. The cell lysate was immunoprecipitated with anti-MYC antibody and then analyzed by Western blotting with anti-MYC and anti-IFT27 antibodies. The cell lysate immunoprecipitated with a mouse normal IgG was used as a control. The anti-MYC antibody pulled down both LZTFL1/MYC and IFT27/GFP. D. Interaction of LZTFL1 with IFT27 in HEK293 cells as determined by G. princeps luciferase complementation assay. HEK293 cells were transfected with the indicated plasmids, and luciferase activity was evaluated 24 h after transfection. The cells expressing both N-Luc-ABHD5 and PLIN1-C-Luc was a positive control. Like the positive control, cells expressing both N-Luc/LZTFL1 and IFT27/C-Luc reconstituted activity. * P<0.01 compared to the N-Luc-LZTFL1 or IFT27-C-Luc only.