Effects of SLF on pp125^FAK tyrosine phosphorylation of pp125^FAK. (A) TF-1 cells were plated on fibronectin-coated wells and incubated for 15 minutes at 37°C. Wells were washed twice with RPMI-BSA medium. The medium with SLF (10 ng/mL) (lanes 2 and 4) or without SLF (lanes 1 and 3) was added to the wells and the wells were further incubated for 10 minutes. Cells were lysed and subjected to immunoprecipitation with anti-pp125^FAK MoAb. The immunoprecipitates were then blotted with anti-p-Tyr MoAb (upper panel). The Western blot transfer membrane that was probed with the anti-p-Tyr MoAb in the upper panel was stripped and probed with the anti-pp125^FAK polyclonal antibody (lower panel). (B) Time course of the SLF-induced enhancement of tyrosine phosphorylation of pp125^FAK. TF-1 cells were plated on the fibronectin-coated well and incubated for 15 minutes at 37°C. Wells were washed twice with RPMI-BSA medium. The medium with 10 ng/mL of SLF was added to the wells. The wells were incubated for various times. Time of incubation is shown in minutes. Cells were lysed and subjected to immunoprecipitation with anti-pp125^FAK MoAb. The immunoprecipitates were then blotted with anti-p-Tyr MoAb (upper panel). The Western blot transfer membrane that was probed with the anti-p-Tyr MoAb in the upper panel was stripped and probed with the anti-pp125^FAK polyclonal antibody (middle panel). Lower graph shows the ratio of tyrosine-phosphorylated pp125^FAK to pp125^FAK protein expression represented by densitometric scanning of transblot bands. Data represent the percentage of control (non-SLF-treated cells) (lower panel). (C) Dose dependence of SLF-induced enhancement of tyrosine phosphorylation of pp125^FAK. TF-1 cells were plated on the fibronectin-coated wells and incubated for 15 minutes at 37°C. Wells were washed twice with RPMI-BSA medium. The medium with increasing concentrations of SLF was added to the wells. Cells were lysed and subjected to immunoprecipitation with anti-pp125^FAK MoAb. The immunoprecipitates were then blotted with anti-p-Tyr MoAb (upper panel). The Western blot transfer membrane that was probed with the anti-p-Tyr MoAb in the upper panel was stripped and probed with the anti-pp125^FAK polyclonal antibody (middle panel). Lower graph shows the ratio of tyrosine-phosphorylated pp125^FAK to pp125^FAK protein expression represented by densitometric scanning of transblot bands. Data represent the percentage of control (SLF nontreated cells) (lower panel). The background band at 50 kD is the immunoglobulin heavy chain from the immunoprecipitation. Similar results were obtained in three independent experiments.