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

Figure 6.

Figure 6

TCR Ligation Does Not Increase pY394-Lck or Lck Kinase Activity

(A) Jurkat cells stimulated for the indicated times with anti-CD3. An aliquot was probed with anti-pY, anti-pY142-ζ, and anti-pY416. Lck (bottom panel) was a loading control.

(B) Human CD4+ T cells were added for the indicated times to unpulsed or sAg-pulsed Raji B cells. Cell activation was detected by pYLAT and pY142-ζ as in (A). Lysates were also probed for pY394-Lck (with anti-pY416) and Lck. The ∼59 kDa species is mostly Fyn.

(C) 2D1 TCR-tg CD8+ T cells were stimulated with either unpulsed or PLP peptide-pulsed LPS-activated B cells from MHC class I HLA-A3 tg mice. Cell activation and pY394-Lck changes were monitored as in (B).

(D) 2D1 TCR-tg CD8+ T cells were stimulated for the indicated times with A3-PLP-MHC class-I tetramers. Cell activation and pY394 changes were detected as in (C). Each of these experiments was performed at least twice.

(E) Confocal IF of purified human CD4+ T cells left untreated or stimulated with anti-CD3 for 2 min at 37°C. Cells were fixed, mixed at a ratio 1:1, and stained with anti-pY416 (top panels) or anti-pY505 (bottom panels), revealed by anti-rabbit Alexa594 (red), and with anti-mouse Alexa488 (green) to detect the presence of anti-CD3. The histograms represent the average anti-pY416 or anti-pY505 fluorescence intensity measurements, in 15 cells, from single-focal planes chosen at random ± SE (13.26 ± 1.4 and 13.59 ± 1.7 n = 15 ROI. p < 0.05) in unstimulated (CD3−) and stimulated (CD3+) cells respectively. The dot plots on the right represent the distribution of fluorescence intensity/cell for the same set of cells. The scale bar represents 5 μm.

(F) Jurkat cells treated for 3 hr with 5 μM Geldanamycin, or DMSO as control, were stimulated for the indicated times with anti-CD3. Cell lysates were probed with anti-pY142-ζ and the intensity of the bands was quantitated by near-infrared fluorescence. Anti-pY142-ζ values, normalized for protein loading (by GAPDH immunoblotting), were plotted against stimulation time so that induction of ζ chain phosphorylation could be designated. The histogram shows the reduction on pY394 and total Lck resulting from geldanamycin treatment. One representative experiment out of three is shown.