FIG 5.

LY6E is associated with CD4 on the plasma membrane and promotes its internalization. (A) Kinetics of CD4 internalization in the presence or absence of LY6E. CD4 internalization was measured by incubating Jurkat or Jurkat-LY6E cells with a PE-Texas Red-conjugated anti-CD4 antibody (clone S3.5; Invitrogen) on ice for 2 h, followed by switching the temperature to 37°C at different periods of time. At each time point, one half of the harvested cells was treated with pH 3.0 in order to remove the surface CD4, the signals thus reflecting the internalized fraction of CD4. The other half was treated with pH 7.0 to measure the total CD4 level. The CD4 internalized rate was calculated as the ratio of pH 3.0-resistant PE-Texas Red fluorescence intensity versus the total cell-associated fluorescence intensity (pH 7.0). Relative rates of internalization were shown by setting the values of parental Jurkat cells at 60 min without acid wash to 1.0. (B) Effect of different chemical inhibitors on the steady-state level of cell surface CD4 expression. DMSO, 80 μM Dynasore, 10 μM CPZ, or 10 mM MBCD was applied to Jurkat or Jurkat-LY6E cells; 8 h after treatment, cells were collected to determine the cell surface expression of CD4 by flow cytometry. The MFI (geometric mean) of each sample was obtained and compared to the value of DMSO-treated parental cells, which was set to 1.0. (C) Lipid raft flotation assay. Jurkat or Jurkat-LY6E cells were lysed and homogenized in 1% Triton X-100 lysis buffer at 4°C, and harvested lysates were subjected to sucrose cushion ultracentrifugation to determine the water-soluble and detergent-resistant membrane fractions. Samples were collected from the top fraction to the bottom fraction upon completion of ultracentrifugation and subjected to SDS-PAGE; CD4/LY6E expression and distribution was determined by immunoblotting using specific antibodies. (D) Analysis of colocalization between LY6E and CD4. Jurkat-TAg cells (Jurkat cells expressing simian virus 40 [SV40] T antigen but with high transfection efficiency) were cotransfected with plasmids encoding CD4, GFP-GPI, or TIM-1. Thirty-six hours after transfection, cells were fixed and costained with specific antibodies. White arrows indicate colocalized areas of the plasma membrane that were positive for both LY6E and CD4. (E) Randomly selected cells (5 fields) were used for colocalization analysis using NIH ImageJ software, and the Pearson’s correlation coefficients were determined and plotted. (F) Analysis of colocalization between LY6E and CD4 in MDMs. MDMs were permeabilized with a permeabilization buffer (Fermentas) and intracellularly stained with Texas Red-conjugated anti-CD4 and anti-LY6E antibodies, followed by incubating cells with a FITC-conjugated anti-rabbit antibody. The nucleus was stained with DAPI. White arrows indicate colocalized LY6E and CD4. Images were collected under 100× magnification and processed through deconvolution software. *, P < 0.05; ***, P < 0.001.