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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: J Recept Signal Transduct Res. 2010 Oct 14;30(6):430–443. doi: 10.3109/10799893.2010.518151

Fig 2. Biochemical analysis of ZAP-70 recruitment and phosphorylation.

Fig 2

A, Immunoprecipitation of TCR complexes from OT-I Rag−/−β2m−/− DP thymocytes stimulated with peptide-loaded APCs. At indicated time points, TCR complexes were immunoprecipitated a monoclonal CD3ε antibody and subjected to Western blot analysis. Co-immunoprecipitated ZAP-70 was probed with monoclonal antibodies against ZAP-70 (first row), p-Y319 (second row) or p-Y493 (third row). Membranes were stripped and reprobed for ζ (third row) as loading control. Representative Western blots are shown from several experiments performed for each peptide (n=3 for OVA, Q4R7 and Q4H7; n=2 for VSV).

B, Graphical evaluation of ZAP-70 recruitment and phosphorylation. Western blots as described in A were quantified by densitometry. Mean grey values of the corresponding bands were background-corrected and normalized to the loading control (ζ) and the values observed at 0min. Graphs show the mean values of several (2–3) experiments performed.