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. 2001 Jan;12(1):1–12. doi: 10.1091/mbc.12.1.1

Figure 3.

Figure 3

Recognition of FAK by phospho-specific antibodies against serine phosphorylated antigens and specific competition by phosphorylated peptides. (A) Reactivity of the phospho-specific antibodies with immune complexes of endogenous FAK (lanes 3 and 4, 7 and 8, 11 and 12, and 15 and 16) and proteins in lysates (lanes 5 and 6, 9 and 10, 13 and 14, and 17 and 18) of unsynchronized (U) and mitotic (M) HeLa cells. FAK is indicated by the arrow; additional cross-reactive bands are indicated by dots (●). (B) Reactivity of the phospho-specific antibodies in Western blots can be specifically blocked by preincubation with phosphorylated synthetic peptide antigen. Each phospho-specific antibody was preincubated at a working concentration of 2-3 μg/ml in the absence of peptide or with a 20- to 100-fold molar excess of nonphosphorylated or phosphorylated peptide. Nitrocellulose strips containing U and M HeLa cell proteins resolved by SDS-PAGE were blocked and then incubated with the pretreated antibody solutions. Total FAK was detected by immunoblotting with the monoclonal antibody 2A7 (lanes 1 and 2). The band corresponding to FAK is indicated by the open arrow; in the anti-pS2 blot, FAK appears as a weak signal migrating above the strong band at ∼120 kDa.