Figure 4.

Src-LAT localizes in the PM outside lipid rafts and is enriched in the IS. A, Mouse CD4⁺ T cells were infected with LATwt-GFP or Src-LAT-GFP (green), stained with anti-CD4 as a PM marker (red) and with DAPI (blue), and then analyzed by fluorescence microscopy (left). An intensity profile for LAT (green) and CD4 (red) along the dashed line was obtained from the merged image (right). The DAPI profile was removed to increase clarity. Frequency scatter plots display the intensity of LAT pixels on the x-axis and the intensity of CD4 pixels on the y-axis. Depicted images are representative of at least 20 GFP⁺ cells each. Bar, 10 μm. B, Transfected J.CaM2 cells (T) were incubated with SEE-pulsed Raji cells (APC) for 20 min. LATwt and Src-LAT were detected by anti-Myc staining. LATwt and Src-LAT enrichment in the IS was observed in >80% of conjugates between J.CaM2 cells and SEE-pulsed Raji cells. The number of conjugates between J.CaM2 cells and unpulsed Raji cells was very low, and enrichment of LATwt or Src-LAT was found in <5% of these conjugates (not shown). Bar, 10 μm. C, Transfected J.CaM2 cells were lysed and subjected to a sucrose gradient fractionation. Fractions were immunoblotted with cholera-toxin B subunit to identify DRM fractions (GM1), and with anti-Myc to detect LATwt and Src-LAT. One representative blot from two independent experiments is shown.