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. 2000 Oct 16;151(2):425–438. doi: 10.1083/jcb.151.2.425

Figure 8.

Figure 8

Effect of carbonate extraction and cholesterol depletion on HA TM mutants. (A) Carbonate extraction. Microsomal membranes were prepared, adjusted to pH 11.0, and HA in the supernatant and pellet fractions was prepared, immunoprecipitated with an anti-HA mAb, and detected as described in Materials and Methods. Processed HA (WT and mutant) was only found in the pellet fraction, indicating that it is not extracted by high pH. (B) Triton X-100 insolubility. CV-1 cells expressing WT or mutant HAs were incubated in the absence (−) or the presence (+) of the cholesterol-depleting reagent MβCD (20 mM) for 30 min at 37°C. HA was prepared and divided into insoluble and soluble fractions and detected as described in Materials and Methods. The percentage of HA found in the insoluble fraction in the absence or presence of MβCD was determined. Cholesterol depletion by treatment with MβCD increases the Triton X-100 solubility of WT HA and GPI-HA, but not of Δ12 HA (n = 4). (C) Effect of cholesterol depletion on fusion. CV-1 cells expressing WT or mutant HAs were prepared as described in the legend to Fig. 4, depleted of cholesterol as described in B, bound to labeled RBCs, and triggered for fusion. Cholesterol depletion by treatment with MβCD does not affect the fusion phenotype of WT HA, GPI-HA, Δ12 HA, or NΔ12 HA (see Fig. 5).