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. 2010 Jun 25;32(6):766–777. doi: 10.1016/j.immuni.2010.05.011

Figure 3.

Figure 3

A Proportion of Lck Is Phosphorylated at Both Y394 and Y505

(A and B) Denatured lysates from (A) Jurkat cells or (B) human CD4+ T cells were treated as in Figures 2A and 2B but immunoprecipitated with anti-pY505 rabbit Ab (pY505) or rabbit IgG (Control). Postdepletion lysates were probed for residual pY505-Lck, Lck, and pY394-Lck. The loading control was ZAP-70. Signal intensity was detected as in Figures 2A and 2B.

(C) Denatured lysates of human CD4+ T cells (left panels) or total mouse thymocytes (right panels) were immunoprecipitated with anti-pY505 or anti-pY416 or rabbit IgG (Control) and immunoblotted with anti-pY416 (red) or anti-pY505 (red), respectively, and with anti-Lck (green). Merged images (yellow) reveal pY394-Lck containing pY505 and vice versa.

(D) Native or denatured Jurkat lysates were immunoprecipitated three times with anti-pY505 or rabbit IgG. Immunoprecipitates were pooled, immunoblotted for pY505, Lck, and pY394-Lck (with anti-pY416), and quantified. These experiments were performed three times with similar results.