Figure 2.

TM4SF5 preferentially binds inactively-closed c-Src forms. (A) Schematic presentation of the human c-Src SH1 domain constructs. (B and C) SNU449 cells stably expressing STrEP-TM4SF5 transiently transfected with either c-Src construct were harvested for whole cell lysates (WCL), and the lysates were precipitated with streptavidin beads, prior to immunoblotting for c-Src. The various c-Src constructs included c-Src WT, SH1, SH1_K298M-HA, and SH1_K298MY530F-HA construct in (B), or SH1, SH1_Y419F, SH1_Y530F, SH1_Y419F/Y530F, SH1_K298M-HA, and SH1_K298M/Y530F-HA constructs in (C). (D) Schematic presentation of the human c-Src SH321 domain constructs. (E and F) SNU449 cells transiently transfected with STrEP-TM4SF5_WT construct together with either c-Src SH321 construct were harvested and precipitated with streptavidin agarose beads, prior to immunoblotting for c-Src, or phospho-Y419 c-Src. The various c-Src SH321 constructs included c-Src SH321, SH321_Y419F, SH321_Y530F, SH321_Y419F/Y530F constructs in (E), or SH321, SH321_Y419F, SH321_{Q531E/P532E/G533I}, and SH321_{Y419F/Q531E/P532E/G533I} constructs in (F). (G) SNU761 cells stably expressing mock or TM4SF5 WT were transiently transfected without (No T/F) or with c-Src WT, c-Src SH432, or c-Src SH1 expression constructs for 48 h prior to invasive ECM degradation analysis for 4 h. Quantitative comparison ratios were calculated from the blots by measurement of band intensities using Image J and normalization of them to those of loading controls. The data shown represent three independent experiments.