Dysregulated Lck expression and phosphorylation and elevated ZAP70 activation in FU-Lck Lckind thymocytes. (A) Western blot analysis showing levels of pTyr in whole thymus lysates taken directly ex vivo from 4 12-week-old Lcktg/Lck−/−/Lckind and 4 age-matched FU-Lcktg/Lck−/−/Lckind mice. Arrows represent molecular mass (kDa) markers. The blot was stripped and reprobed with anti-ERK2 to monitor protein loading. (B) Levels of ZAP70 Tyr319 phosphorylation were assessed; loading control = total ZAP70 expression. (C) Phosphorylation of Lck was assessed using Lck pTyr505 and Src pTyr416 Abs. The Src pTyr416 Ab cross-reacted with Lck pTyr394 in T cells. The ERK2 western blot serves as a loading control. (D) Expression of Lck and FU-Lck transgenes in thymocyte lysates was determined by Western blot analysis using a V5 mAb. Parallel blots using an Lck mAb monitored total Lck expression, while ERK blots functioned as loading controls. For all blots, images were obtained using an infrared imaging system (LI-COR). (E) FACS histogram showing intracellular levels of Lck in FU-Lcktg/Lckind and Lckind thymocytes. Cells were permeabilized with 0.5% saponin before Ab staining. (F) Levels of intracellular ZAP70 pY319 in gated DP thymocytes from FU-Lcktg/Lckind and Lckind mice. Cells were stained for surface CD4 and CD8 before fixation in 2% formaldehyde, permeabilization in 90% methanol, and ZAP70 pY319 Ab staining. Histograms represent 1 of 3 repeated experiments.
