Fig. 5. Binding of pTyr by PKCθ is required for early TCR signaling.

(A to D) GFP⁺ primary CD4⁺ T cells were isolated from BM chimeric mice reconstituted with PKCθ knockout BM cells transduced with RV encoding GFP and either PKCθ^WT or PKCθ^HR2A (A to C) or empty vector (C). The cells were left unstimulated or stimulated with mAbs against CD3 and CD28 for 5 min before cell lysates were subjected to SDS-PAGE and immunoblotted for the indicated proteins. p-Lck and p-TCRζ were determined by the dominant lanes of molecular weight around 55 kDa (Lck) and 23 kDa (TCRζ) from the total pTyr blot (4G10), respectively. Representative raw data are shown in (A) and (C) and pooled data from 3 independent experiments were quantitated by densitometry in (B) and (D). ns, not significant, *P < 0.05, **P < 0.01.

(E) Cells isolated and stimulated as in (A) and (C) were fixed and stained intracellularly with an APC-conjugated antibody against phosphorylated Itk (p-Itk). Plot is representative of 3 independent experiments.