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. 2010 Jun 25;32(6):766–777. doi: 10.1016/j.immuni.2010.05.011

Figure 1.

Figure 1

Activated SFKs in T Cells and Thymocytes

(A) Dual fluorescence immunoblot (IB) for Lck (green) and pY416 (red) in Jurkat cells, TCR-, CD4-, and CD8-deficient 31.13 (TCR-) and TCR-reconstituted 31.13 (TCR+) Jurkat cells, human and mouse CD4+ and CD8+ T cells, and mouse thymocytes from normal and Rag1−/− mice. Merged (yellow) denotes expression of pY394-Lck (see Figure S1C). The ∼59 kDa species is mostly pY418-Fyn.

(B) Confocal IF of human CD4+ T cells stained for CD4 and either for Lck or active SFKs (pY416). Nuclei were counterstained with Hoechst 33258 (blue). The scale bar represents 3.5 μm.

(C) Confocal IF for Lck forms in human CD4+ T cells. Double staining with two different Abs to Lck, a rabbit Ab (red) and a mouse mAb (green) (first row), was used as the positive control for colocalization. Double staining with a rabbit Ab to Lck (green) and a mAb to Filamin A (red) (bottom row) was the negative control (lack of colocalization). The presence of pY394-Lck was identified by double staining with Lck mAb (green) and pY416 rabbit Ab (red) (second row). For pY394 and pY505 colocalization (third row), we used anti-pY416 rabbit Ab (green) and anti-pY505 mAb (red). The degree of colocalization was determined with Imaris colocalization software. Corresponding pictures were merged (third column) and all pixels colocalized were represented as white spots (fourth column). Each colocalized pixel was plotted on a scatter diagram for producing correlation plots with colocalizing pixels falling around the diagonal line (right-hand column). Data from at least 50 cells for each condition were used for calculating the Pearson's correlation coefficient (Rr) as mean ± SE p values (two-tailed) determined with Student's t test. The scale bar represents 3.5 μm.

(D) Confocal IF images of a mouse spleen, LN, and thymus. Tissue sections (10 μm) were stained with biotin-labeled anti-mouse CD4 (L3T4) (red) and anti-pY416 (rabbit) (green). The scale bar represents 10 μm. Images are representative of three independent experiments.