Figure 1.

ANGII stimulates FAK phosphorylation at Ser-910 in IEC-18 cells. (A). Knockdown FAK expression by siRNA inhibits ANGII induced-cell migration in IEC-18 cells. IEC-18 cells were transfected with Trans IT-TKO alone, non-targeting negative control or 75nM FAK siRNA. After 48 h cells were wounded as described in Materials and Methods and incubated with or without 50nM ANGII for 16h. Experiments were terminated by washing cells twice in PBS, followed by fixing in 10% buffered formalin phosphate for 20 min. Cells were stained with Wright-Giemsa and observed under phase contrast with a ×10 lens mounted on an upright microscope. Upper Panel: Photographs representative of control, non-targeting siRNA or FAKsiRNA transfectd cells. Results are typical of five independent experiments. Lower panel: Values are means ± SE of at least 8 fields from five independent experiments and are expressed as the number of cells observed across the wound margin at 16 h. * P < 0.01 vs. control, ** P<0.01 vs. control with ANGII treatment. (B). Time-course of ANG II in stimulation of FAK phosphorylation at Ser-910 in IEC-18 cells. Upper Panel: Confluent and quiescent IEC-18 cells were treated at 37°C with 50nM of ANG II for various times as indicated and subsequently lysed in 2×sample buffer. FAK phosphorylation at Ser-910 or Tyr-397 was analyzed by Western blotting with pSer-910 Ab or pTyr-397 Ab. The membranes were further analyzed by Western blotting using anti-FAK Ab. The autoradiograms shown are representative of at least three independent experiments. Lower Panel: Quantification of FAK phosphorylation at Ser-910 and Tyr-397 was performed by scanning densitometry. Values shown as bars are the mean of at least three independent experiments and are expressed as the percentage of the maximal increase of phosphorylation above control (unstimulated) values.