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. Author manuscript; available in PMC: 2014 Jun 1.
Published in final edited form as: Traffic. 2013 Mar 20;14(6):650–662. doi: 10.1111/tra.12064

Figure 6. Regulatory lipids affect trans-SNARE complex formation and interactions with the HOPS complex.

Figure 6

16X standard fusion reactions containing 48 μg of BJ3505 CBP-Vam3p nyv1Δ vacuoles and 48 μg DK6281 vacuoles were treated with inhibitory concentrations of the following lipid binding domains: 2 μM GST-FYVE, 25 μM GST-PX, 1 μM MBP-FappPH, 10 μM Ent3p-ENTH, 10 μM Epsin1-ENTH, 10 μM GST-C1b, 2 μM His6-SigD or 20 μM filipin for 90 minutes at 27°C. (A) Samples (30 μl) were withdrawn from each reaction to measure Pho8p activity following fusion. Error bars represent SEM (n=3). (B) The reaction remainders were centrifuged to isolate the vacuoles. Membranes were then solubilized and CBP-Vam3p complexes were affinity purified using calmodulin agarose as described in the Materials and Methods. Trans-SNARE complexes were detected by immunoblotting for Vam3p, Nyv1p and Vam7p. The Image shown is representative of 3 independent trials.