Fig. 7.

Downregulation of CD4 is dependent on Tip interaction with Vps35. (A) Multicolor flow cytometric analysis of Jurkat stable T cell lines expressing vector, wt Tip, or Tip Δ24 or Tip E/Q mutant with antibodies against TCR (top), CD4 (middle), and CD45 (bottom). (B) Western blot analysis of whole-cell lysates from the indicated Jurkat stable cell lines, probing for CD4 and β-tubulin (as a loading control). (C) Quantitative real-time PCR analysis of CD4 mRNAs. For the relative quantification of CD4 mRNAs, 18S rRNA was used for normalization. (D) Confocal microscopy of Jurkat T cells transiently expressing CD4 (red) and vector (top), wt Tip (green; middle), or the Tip Δ24 mutant (green; bottom). (E) Recovery of CD4 surface expression after trypsin treatment. Jurkat stable T cell lines expressing vector, wt Tip, or Tip Δ2, Tip Δ24, or Tip E/Q mutant were treated with trypsin to remove cell surface-associated CD4, washed, and incubated at 37°C with RPMI 1640. Cells were harvested at the indicated time points and stained with anti-CD4 antibody, followed by flow cytometric analysis.