FIG. 4.

Effects of Cys-to-Ser mutations on lipid raft localization of the Env protein. (A, B, C) HeLa cells were transfected with either pHCMV-VSV G or pCDNA3-CD4 (A), WT pHXB2RU3 (B), or each of the WT and Cys-to-Ser mutant pHXB2RI3 proviruses (C). Cell lysates prepared by 1% Triton X-100 extraction at 4°C were subjected to sucrose gradient equilibrium ultracentrifugation. After fractionation, proteins in each fraction were analyzed by Western blotting using VSV G or SIM2 MAbs (A); MAbs 902, Chessie 8, and 183 (B); and MAb Chessie 8 (C). The distribution of gp41 in sucrose gradients is shown in panel C. (D and E) 293T cells were cotransfected with each of the WT or mutant pSVE7puro plasmids along with pIIIextat. Cell lysates prepared by 1% cold Triton X-100 extraction were subjected to sucrose gradient equilibrium ultracentrifugation and analyzed by Western blotting using Chessie 8 (D). Soluble fractions 1 to 3 and raft-associated fractions 7 and 8 were separately combined and normalized for protein concentrations. Equal amounts of proteins from soluble (S) and raft (R) fractions were subjected to Western blotting using MAb Chessie 8, and the gp41 band is shown (E). The distribution of WT and mutant gp41 in soluble and raft fractions was quantified as described in Materials and Methods.