Figure 3.

CD3ɛ-PRS mediates CD3 tonic ubiquitylation in DP thymocytes. (A) Thymi were isolated from WT or CD3e^ΔPRS/ΔPRS mice. Surface expression of the TCR:CD3 complex was measured by flow cytometry on thymocytes co-stained with mAbs against CD4, CD8, and TCRβ. (B, C) Thymocytes were stained with anti-CD4 and anti-CD8, and DP and SP thymocytes were purified by FACS. Sorted thymocytes were fixed, permeabilized, and stained with Alexa-647-conjugated CD3ζ mAb. The distribution of CD3ζ in thymocyte populations is indicated (n>50) (B). The localization of CD3ζ (red) and DAPI-stained nucleus (blue) are shown in representative confocal images (C). (D) Tonic ubiquitylation of CD3 was performed as described in Figure 1A.