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Figure 8


Fig. 8. Association with TERM is essential for the pro-migratory activity of L6-Ag. (A) Predicted cytoplasmic regions are involved in recruitment of L6-Ag to TERM. 293T cells were transiently transfected with the plasmids encoding wild-type L6-Ag or various mutants of L6-Ag. After 48 hours cells were lysed in 0.8% Brij 98/0.2% Triton X-100 and immunoprecipitation was carried out using the anti-L6-Ag mAb (odd lanes) or a negative control mAb (even lanes). Proteins were resolved in 11% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with either anti-CD151 polyclonal Ab or the anti-L6-Ag mAb (L6pke). (B) Syntenin-1 is not involved in the recruitment of L6-Ag to TERM. MDA-MB-231 cells were transiently transfected with either control siRNA (si-Cont) or siRNA that targets syntenin-1 (si-Syn1). After 72 hours cells were lysed in 0.8% Brij 98/0.2% Triton X-100 and immunoprecipitation was carried out using the anti-L6-Ag mAb. Proteins were resolved in 11% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with either anti-CD151 polyclonal Ab or the anti-L6-Ag mAb (L6pke). The degree of syntenin-1 knock-down was assessed by analysing total cell lysates with anti-syntenin-1 mAb (lanes 1 and 2). (C) Effect of L6-Ag mutations on the pro-migratory activity of the protein. MCF-7 cells were co-transfected with plasmids encoding GFP and various L6-Ag constructs. Migration experiments towards fibronectin were performed 48 hours after transfection. Migration was quantified by counting cells in seven random fields per membrane (~10-20 cells/field) as described in detail in Materials and Methods. Data are reported as fold increases over migration of cells transfected with the control plasmid (GFP). Data (all graph panels) are shown as mean ± s.d. calculated from at least three separate experiments each performed in triplicate. P values were calculated using the two-tailed t-test. L6wt, wild-type L6-Ag.





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