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. 2018 Jul 23;178(1):13–17. doi: 10.1104/pp.18.00577

Figure 2.

Figure 2.

The GPS system is suitable for analyzing protein-protein interactions of the soluble SEC11Δ149. A, Diploid yeast expressing OST4pSEC11-CubPLV or OST4pSEC11Δ149-CubPLV as bait and a NubG-X fusion of SYP121 and the controls (negative, NubG; positive, NubI) as prey were spotted onto different media as indicated. Growth on CSM−LTUM was used to verify the presence of both bait and prey vectors in the diploid yeast. Growth on CSM−LTUMAH was used to verify interaction, and additions of different concentrations of Met were used to suppress bait expression as a test for interaction strength. Diploid yeast was dropped at 1.0 and 0.1 OD600 in each case. Incubation time was 24 h on CSM−LTUM and 72 h on CSM−LTUMAH. B, Diploid yeast expressing Exg2 GPI signal peptide-fused Exg2pSEC11-CubPLV and Exg2pGPI-SEC11Δ149-CubPLV as bait, with NubG-SYP121 fusion and the controls (negative, NubG; positive, NubI) as prey spotted onto different media as indicated. Growth was as in A. C, Immunoblot analysis of the diploid yeast carried out with commercial anti-HA antibody for the prey fusions and anti-VP16 antibody for the bait fusions. Ponceau S stains were used for blotting/loading control.