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. Author manuscript; available in PMC: 2015 Oct 9.
Published in final edited form as: Cell. 2014 Oct 2;159(2):333–345. doi: 10.1016/j.cell.2014.08.042

Figure 2. Quantitative determination of Lck coupling to CD4, CD8, and CD8.4 coreceptors.

Figure 2

Cell lysates were incubated with beads coated with antibodies to CD4, CD8β, or isotype controls. Beads were probed with PE-conjugated antibodies to Lck, CD8α, or CD4 and analyzed by flow cytometry. (A-B) Sorted DP CD3low thymocytes from WT mice were analyzed. (C-D) Thymocytes from CD8WT and CD8.4 OTI Rag2−/− β2m−/− mice were analyzed. Representative histograms (A, C) and aggregate data (B, D) (mean ± SD, n = 3-5) are shown. P values were calculated using Student's T test (2 tailed, unequal variance). See also Figure S2. (E) Lck was immunoprecipitated from lysates from nontreated (NT), pervanadate (PV), or 20 μM PP2 treated CD8WT and CD8.4 OTI Rag2−/− β2m−/− thymocytes. Phosphorylation of Lck was analyzed by Western blotting using simultaneous staining with Abs specific for phosphorylated or non-phosphorylated Y394. The membrane was re-probed with Ab to total Lck. Percentage of phosphorylated Lck molecules in resting CD8WT or CD8.4 DP thymocytes was calculated. CD8WT: n=4; CD8.4: n=5.