The anti-Kar2 rabbit polyclonal antibody was a gift from E. Miller (Medical Research Council Laboratory of Molecular Biology, Cambridge, England, UK) and was used at a 1:10,000 dilution. The anti-GFP mouse monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (sc-9996) and was used at a dilution of 1:500. The anti–glucose-6-phosphate dehydrogenase (G6PDH) rabbit polyclonal antibody was purchased from Sigma-Aldrich (SAB2100871) and was used at a concentration of 1:30,000.
To test whether Sec7 activity plays a role in TRAPPIII recruitment to Golgi compartments, 40 µM 6-methyl-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one (MNTC; MolPort) was added to growth media 20 min before imaging. To deplete TRAPPIII from the Golgi in anchor-away experiments, cells expressing Pma1-2×FKBP and Trs85-mRFPmars-FRB were treated with 1 µg/ml rapamycin (LC Laboratories) for 1 h before imaging. The rapamycin growth plates shown in Fig. S2 D contained 1 µg/ml rapamycin.
To test whether Sec7 activity plays a role in TRAPPIII recruitment to Golgi compartments, 40 µM 6-methyl-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one (MNTC; MolPort) was added to growth media 20 min before imaging. To deplete TRAPPIII from the Golgi in anchor-away experiments, cells expressing Pma1-2×FKBP and Trs85-mRFPmars-FRB were treated with 1 µg/ml rapamycin (LC Laboratories) for 1 h before imaging. The rapamycin growth plates shown in Fig. S2 D contained 1 µg/ml rapamycin.