Figure 5.

The α₄(Y991A)β₁ mutant poorly associates with talin. (A) The α₄(Y991A)β₁ complex does not properly recruit talin. Total lysates (left) or talin coprecipitating with anti-α₄, anti-β₁, or an irrelevant mouse IgG (right) from lysates of either JB4 transfected with wt α₄ or the α₄(Y991A) mutant (top). The blot was stripped and reprobed for α₄ (bottom). (B) Silencing of talin in JB4 cells expressing either wt or α₄(Y991A). The indicated cells were transfected with either talin1-specific or control siRNA. Total lysates of each group were immunoblotted with talin or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 66 and 67% in talin content in JB4 expressing either wt or α₄(Y991A), respectively. (C) Talin suppression preferentially impairs wt α₄β₁–mediated resistance to detachment from sVCAM-1 (2,220 sites/μm²). *, P < 0.03 for control compared with talin-silenced cells at 0.5 dyn/cm². Where indicated, cells were pretreated with the α₄β₁-specific blocker BIO1211. (inset) Effect of talin suppression on resistance to detachment from VCAM-1–Fc (30 CAM sites/μm²) of JB4 cells expressing either wt or α₄(Y991A). In each panel, the mean ± range of two experimental fields is depicted. Results are representative of three independent experiments. Error bars represent SD.