Skip to main content
. 2003 Aug;77(16):9041–9051. doi: 10.1128/JVI.77.16.9041-9051.2003

FIG. 4.

FIG. 4.

Requirement of Tip-p80 interaction for localization in lipid rafts. (A) Effect of Tip expression on p80 localization in lipid rafts. 293T cells were transfected with an expression vector containing the Xpress-tagged p80 alone (top two panels) or together with the AU1-tagged wild-type Tip (middle three panels) or the Tip Δ2 mutant (bottom three panels). At 48 h posttransfection, cells were lysed with 1% Triton X-100 and subjected to discontinuous centrifugation. Each fraction was subjected to immunoblot assay with anti-Xpress or anti-AU1 antibody. Horseradish peroxidase-conjugated CT-B (GM1) was used as a control to locate the lipid raft fractions. Fractions 3 to 5 and 9 to 12 contained the raft region and the soluble region of the gradients, respectively. Based on densitometry analysis with the TINA software program, the lipid raft fractions and soluble fractions, respectively, contained 5% and 83% (top panel), 49% and 19% (middle panel), and 4% and 84% (bottom panel) of p80, 65% and 6% (middle panel) and 41% and 20% (bottom panel) of Tip, and 79% and 11% (top panel), 83% and 6% (middle panel), and 78% and 21% (bottom panel) of GM1. (B) Requirement for p80 interaction but not Lck interaction for lipid raft localization of Tip. 293T cells were transfected with an expression vector containing the AU1-tagged wild-type Tip (top two panels), Tip Δ2 (second two panels), or Tip mSH3B (third two panels). Cells were also cotransfected with Tip and Lck expression vectors (bottom three panels). At 48 h posttransfection, cells were lysed with 1% Triton X-100 and subjected to discontinuous centrifugation. Each fraction was subjected to the immunoblot assay with anti-AU1 antibody or anti-Lck antibody. Horseradish peroxidase-conjugated CT-B (GM1) was used as a control to locate the lipid rafts. Based on densitometry analysis with the TINA software program, the lipid raft fractions and soluble fractions, respectively, contained 72% and 5% (top panel), 31% and 52% (second panel), 53% and 9% (third panel), and 71% and 10% (bottom panel) of Tip and 79% and 7% (top panel), 87% and 4% (second panel), 82% and 7% (third panel), and 87% and 4% (bottom panel) of GM1, and 47% and 46% (bottom panel) of Lck.