Fig 1: Validation of putative effectors for Rab2A, Rab5A and Rab9A.(A) Affinity chromatography of cell lysates (input) using GST-fusions to the indicated GTP-locked (QL) or GDP-locked Rabs. The bound proteins were eluted and probed with antibodies to the indicated proteins. Lysates were from HEK293 cells and probed for STAMBPL1, or from rat brain and probed for ARFGEF3. (B) Confocal micrographs of MEFs expressing either GFP-STAMPBL1 alone and probed for a Golgi marker, or co-expressing the mitochondrial Rab2A(Q65L)-BirA* as indicated. (C) As (B) except that the cells were expressing GFP-ARFGEF3. (D) Coomassie blue stained gel showing affinity chromatography of purified recombinant GFP-STAMBPL1 using GST fusions to the indicate GTP-locked (QL) or GDP-locked (SN) Rabs. The recombinant GFP-STAMBPL1 binds specifically to the GTP form of Rab2A. (E) Affinity chromatography of HeLa cell lysates (input) using GST-fusions to the indicated GTP-locked (QL) or GDP-locked Rabs. The bound proteins were eluted and probed with antibodies to the indicated proteins. TBCK, OSBPL9 and RELCH show specific binding to the GTP form of Rab5, with RELCH also binding to Rab11-GTP as previously reported (Sobajima et al., 2018). (F) Confocal micrographs of cells expressing GFP-TBCK alone, or with co-expression of mitochondrial Rab5A(Q79L)-BirA* as indicated. (G) Coomassie blue stained gel showing affinity chromatography of lysate from E. coli expressing His6-Nde1 (input) using GST fusions to GTP-locked (Q66L) or GDP-locked (S21N) Rab9A. The samples were also immunoblotted for the His6 tag. The recombinant His6-Nde1 binds specifically to the GTP form of Rab9A. (H) Affinity chromatography of lysate from HEK293 cells expressing FLAG-HPS3 (Input) using GST-fusions to GTP-locked (QL) or GDP-locked (SN) Rab9A. The bound proteins were eluted and probed for the FLAG tag. (I) Confocal micrographs of HeLa cells expressing FLAG-HPS3 and mitochondrial Rab9A(Q66L)-BirA* (GTP-locked) or Rab9A(S21N)-BirA* (GDP-locked). The GTP-form specifically recruits HPS3 to mitochondria. (J) Affinity chromatography of HEK293 cell lysate (Input) using GTP-locked (QL) or GDP-locked (SN) GST-Rab11A. Bound proteins were eluted and probed for ALS2/alsin.