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. Author manuscript; available in PMC: 2014 Jul 21.

Published in final edited form as: Virology. 2010 Sep 28;408(1):89–102. doi: 10.1016/j.virol.2010.08.031

Vpu fusion proteins also partition into detergent resistant membranes and cholesterol depletion reduces the amount of VpuEGFP and Vpu_SCEGFP1 in DRM fractions. Left side of panels A and B. 293 cells were transfected with vectors expressing either VpuEGFP (panel A) or Vpu_SCEGFP1 (panel B) proteins. At 48 hours, cells were lysed in ice cold DRM buffer containing 1% Triton X-100, and raft proteins separated from non-raft proteins on discontinuous sucrose gradients by ultracentrifugation as described in the Materials and Methods section. Fractions 1-3 (I), fractions 6-7 (M) and 10-12 (S) were pooled, concentrated and the Vpu proteins detected by Western blot analysis using a rabbit anti-EGFP serum. Right side of panels A and B. 293 cells were transfected with vectors expressing either VpuEGFP (Panel A) or Vpu_SCEGFP1 (Panel B) proteins. Following transfection, cells were incubated in the presence of 4 μM lovastatin for 48 hours. Thirty minutes prior to lysis, M-β-CD was added to a final concentration of 10mg/ml. Cells were processed as described for the untreated samples. Panels C-D. Micrographs of untreated cultures (Panel C) or those treated with M-β-CD (Panel D).

Vpu fusion proteins also partition into detergent resistant membranes and cholesterol depletion reduces the amount of VpuEGFP and Vpu_SCEGFP1 in DRM fractions. Left side of panels A and B. 293 cells were transfected with vectors expressing either VpuEGFP (panel A) or Vpu_SCEGFP1 (panel B) proteins. At 48 hours, cells were lysed in ice cold DRM buffer containing 1% Triton X-100, and raft proteins separated from non-raft proteins on discontinuous sucrose gradients by ultracentrifugation as described in the Materials and Methods section. Fractions 1-3 (I), fractions 6-7 (M) and 10-12 (S) were pooled, concentrated and the Vpu proteins detected by Western blot analysis using a rabbit anti-EGFP serum. Right side of panels A and B. 293 cells were transfected with vectors expressing either VpuEGFP (Panel A) or Vpu_SCEGFP1 (Panel B) proteins. Following transfection, cells were incubated in the presence of 4 μM lovastatin for 48 hours. Thirty minutes prior to lysis, M-β-CD was added to a final concentration of 10mg/ml. Cells were processed as described for the untreated samples. Panels C-D. Micrographs of untreated cultures (Panel C) or those treated with M-β-CD (Panel D).