The Rab GEFs of the Drosophila Golgi apparatus. (A) Cartoon of the SBP-GFP TAP tag and the strategy used to make stable cell lines or fly lines by CRISPR and then analyzing the tagged proteins in cell lines or flies. AC, affinity chromatography; MS/MS, tandem mass spectrometry. (B) Widefield micrographs of cells transiently transfected with TAP-tagged Golgi GEFs and labeled with antibodies to the indicated Golgi markers. Images are representative of at least two independent experiments, with at least three micrographs obtained from each. Bars, 5 µm. (C) Immunoblot of stable cell lines expressing SBP-GFP–tagged TRAPPC3 or Rgp1. (D) TAP of TRAPPC3 from stably transfected cell lines. The purification was on anti-GFP beads with elution by TEV protease and then on streptavidin beads with elution by biotin. The starting tagged protein (arrows) and the final purified protein (arrowhead) are indicated. FT, flowthrough. (E) Silver-stained protein gel comparing single-step affinity chromatography of TRAPPC3–SBP-GFP using the GFP tag (TEV eluate) with TAP using both the GFP and the SBP tag (biotin eluate). Molecular masses are given in kilodaltons. (F) Mass spectrometry analysis of TAPs of SBP-GFP–tagged TRAPPC3 and Rgp1. For each protein that was identified by mass spectrometry, the total spectral counts are shown for the TRAPPC3 or Rgp1 purifications. Duplicates are shown. CG, computed gene; Cont., untransfected cells.