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. 2000 Mar 20;148(6):1231–1238. doi: 10.1083/jcb.148.6.1231

Figure 1.

Figure 1

Physical associations of Vam2p and Vam6p. (A) Vam2p and Vam6p form a stable complex. BJ3505 HA-Vam6p vacuoles were solubilized with buffer containing 1% Triton X-100 or incubated at 27°C in a complete fusion reaction containing Gdi1p (64 μg/ml) for 90 min. Detergent extracts and release reaction supernatants were immunoprecipitated with antibody against Vam2p or preimmune antibody as described in Materials and Methods. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with anti-HA mAb 12CA5. (B) Sucrose velocity gradients. BJ3505 vacuoles (500 μg) were solubilized in 400 μl of PS solubilization buffer and analyzed on 10–40% sucrose gradients as described in Materials and Methods. Gradient fractions were analyzed by SDS-PAGE and immunoblotting with antisera against Vam2p, Vam6p, Nyv1p, and Vam3p. Immunoblots were then quantified by scanning densitometry and plotted as a percent of the maximum signal in the peak fraction.