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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Traffic. 2016 Jul 24;17(10):1091–1109. doi: 10.1111/tra.12423

Figure 5. Vacuoles from a pah1Δ deletion strain have a decrease in the level of Sec18p bound to cis-SNARE complexes.

Figure 5

Vacuoles were harvested from BJ3505, RFY17 (BJ3505 pah1Δ), and RFY19 (BJ3505 pah1Δ, pPAH1) strains and assayed for Sec18p binding to cis-SNARE complexes. (A) Recombinant GST-Vam7p was added to fusion reactions and incubated at 27°C for 30 min in the absence of ATP to allow formation of cis-SNARE complexes containing GST-Vam7p. Next, ATP regenerating system was added and the samples were incubated at 27°C for the indicated times before vacuoles were isolated by centrifugation and solubilized. Glutathione Sepharose was used to pull down GST-Vam7p and attached proteins were resolved by SDS-PAGE and imaged by Western blot. (B) WT versus pah1Δ vacuoles. (C) WT versus pah1Δ + pPAH1 vacuoles. (D) WT versus pah1Δ vacuoles + 200 µg/ml rPah1p. Densitometry values for Sec18p pull down were normalized against the corresponding pull down Vam7p value. Graph shows the normalized average ratios (n=3). The white bars represent the pah1Δ pulldown data from panel B. This is to facilitate visualization of the effect of adding recombinant Pah1. * P<0.05; ** P<0.001.