Research Paper Volume 14, Issue 7 pp 2989—3029

The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network


Figure 4. Interaction partners of NtFT4 identified in immunoprecipitated protein complexes after transient expression in S2 cells. The abundance of the interaction partners was confirmed by immunodetection using mouse anti-Myc (top) or rabbit anti-HA (bottom) antibodies in the extracts and successful precipitation with magnetic anti HA-beads was confirmed by the detection of HA-EGFP-NtFT4 in the eluates. (A) Western blots of extracts (Input) and eluates after co-immunoprecipitation (Eluate) following transient co-transfection of S2 cells with HA-EGFP-NtFT4 plus Myc-Tsn, Myc-14-3-3 ζ, Myc-CG4364, Myc-Df31, Myc-Rack1, Myc-CCT7, Myc-PyK, Myc-CCT2, Myc-Hsp26, Myc-Pen, Myc-Cbs or Myc-p47. Detection of co-immunoprecipitated proteins in the eluate is indicated by red arrowheads. Df31 was not detected in extracts under the mild conditions used for immunoprecipitation (empty arrowhead). (B) Analysis of FRET efficiency in co-transfected cells expressing the donors Cerulean (Cer, negative control), Cer-NtFT4 or Cer-CG7054 plus the acceptors EYFP-CCT7, EYFP-CG4364, EYFP-Df31, EYFP-Hsp26, EXFP-p47, EYFP-Pen, EYFP-PyK or EYFP-Tsn by flow cytometry. Gating strategy and representative controls are shown in Supplementary Figure 8. Cer-NtFT4 and Cer-CG7054 were co-transfected in three independent triplicates (n = 3) and statistical significance was tested by one-sample t-test (****p < 0.001, ***p < 0.01, **p < 0.05, *p < 0.1, Abbreviation: NS: not significant).