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Fig. 1. Localization of LATWT-, LAT ${Delta}$ 32-104-GFP and LAT ${Delta}$ Ct-GFP in T cells. (A) Schematic representation of the mouse LAT constructs. Domains include transmembrane (TM), palmitoylated cysteine residues (CC), green fluorescent protein (GFP) and tyrosine (Y) residues. (B) Jurkat cells expressing LATWT-GFP, LAT ${Delta}$ 32-104-GFP or LAT ${Delta}$ Ct-GFP were pulsed with transferrin coupled to cyanine 3 (Tf) and labelled either with the mAb CTR433, which stains the cis-Golgi, or an anti- ${zeta}$ mAb. Shown are single-colour or two-colour overlay images acquired with a confocal microscope at a medial Z-section. Insets display 3x enlargement of details comprised in the white box. White arrows point to co-localization areas and white arrowheads show vesicles where only Tf is found. Bar, 5 µm. (C) GFP fluorescence intensity of the intracellular and plasma membrane pools of LAT was measured on 12-bit confocal images. Percentage of intracellular versus total LAT is plotted for LATWT-GFP- ( ${bullet}$ , n=29), LAT ${Delta}$ Ct-GFP- ( ${circ}$ , n=33) and LAT ${Delta}$ 32-104-GFP- ( ${square}$ , n=28) expressing cells. Shown are mean±s.d., which are significantly different (P<0,0001, unpaired Student's t-test). (D) T-cell blasts were fixed and co-labelled with anti-LAT (left panel) and anti- ${zeta}$ (right panel) Abs. Images show the presence of endogeneous intracellular pools of LAT and ${zeta}$ . Bar, 5 µm.

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