Figure 1. Selective dysregulation of Lck phosphorylation in E613R T cells.

(A) Whole-cell lysates of resting LN CD4 T cells from +/+, H/H, and E613R mice were blotted with Ab to the inhibitory and activating tyrosines of Lck (Lck pY505/Src pY416). Src416 Ab binds activating tyrosines of all SFKs. The lower band represents p56 Lck; the upper band, p59 Fyn. Densitometry was performed on the lower Lck band. Total Lck is detected as a loading controls.

(B) Whole-cell lysates of resting thymocytes from +/+, H/H, and E613R mice were blotted with Ab to the inhibitory and activating tyrosines of Lck (Lck pY505/Src pY416). The single band detected by Src416 in thymocytes represents Lck. Total Lck is detected as a loading control.

(C) Fyn was immunoprecipitated from whole-cell lysates of resting thymocytes from +/+, H/H, and E613R mice. Immunoprecipitates (IPs) were subsequently blotted with Ab to the inhibitory and activating tyrosines of Fyn (pSrc527/pSrc416). Total Fyn is detected as loading control.

(D) TCR-associated ζ chain was immunoprecipitated from whole-cell lysates of resting thymocytes from CD45 allelic series and E613R mice. IPs were subsequently blotted for total ζ and total phosphotyrosine (pY) levels.

Data in 1A–D are representative of at least two independent experiments.

(E) TCR-stimulated, fixed, and permeabilized thymocytes were stained for phospho-Erk and costained for CD4 and CD8 so that DP subsets could be identified. Data were collected by flow cytometry. Histograms depict intracellular phospho-Erk in DP subsets from allelic series and E613R thymi. Data are representative of at least three independent experiments.

Densitometry measurements in this and all subsequent figures were normalized to wild type.