Analysis of β2-phosphorylation status in stimulated T cells and peptide affinity chromatography with phosphorylated and nonphosphorylated β2 cytoplasmic peptides. (A) β2 integrins are phosphorylated on Thr758 in T cells after phorbol ester and TCR ligation. Isolated human T cells were either nonstimulated (C) or preincubated with okadaic acid (OA) and stimulated with PDBu or OKT3. The cells were lysed and extracts subjected to Western blotting with the pThr758-specific antibody (pT-β2) or with the R2E7B antibody (β2). Vertical lines have been inserted to indicate a repositioned gel lane. (B) Binding of filamin and 14-3-3 to a phosphorylated control peptide (pαL), β2-35 (WT), β2-35A (T/A), and β2-35pT (PT) peptide affinity columns. The lysates and the bound materials were analyzed by Western blotting with α-actinin, 14-3-3, and filamin antibodies. Vertical lines have been inserted to indicate a repositioned gel lane. (C) Binding of purified talin head domain and 14-3-3ζ to peptide affinity columns. Talin head fragment (44 nM) binds to all β2 integrin peptides (WT, T/A, PT) in a similar way, thus indicating that talin binding is independent of β2 integrin Thr758. The control peptide (pαL) binding is much weaker, which implies specific binding. The binding of talin to PT is completely outcompeted when a similar molar amount of 14-3-3ζ was added in the incubation volume. The concentrations of the 14-3-3 added were 36 nM (S1), 71 nM (S2), and 143 nM (S3), and 44 nM talin was present in each column.