Fluid flow magnitude-dependent Lyn-Src activity.

<p>(A) Selective Lyn-Src activities in response to different magnitudes of fluid flow. <i>n</i>>7 cells. (B) Cyto-Src activity is not altered by fluid flow. <i>n</i>>7 cells. (C, D) C28/I2 cells were cotransfected with either Lyn-Src or Cyto-Src biosensor and eIF2α siRNA or NC siRNA, and then subjected to fluid flow (10 dynes/cm²) during FRET imaging. (C) Lyn-Src activity under fluid flow. <i>n</i>>7 cells. (D) Cyto-Src activity under fluid flow. <i>n</i>>7 cells. (E) Lyn-Src activity in cytokine-treated cells under fluid flow(5 dynes/cm²). Cells transfected with a Lyn-Src biosensor were pretreated with cytokines for 2 hour before FRET imaging. <i>n</i>>7 cells. Scale bars, 10 µm.</p

Salubrinal (Sal) and guanabenze (Gu) increase phosphorylation of eIF2α and decrease Cyto-Src activities.

<p>(A) Western blots showing the elevated level of p-eIF2α by salubrinal and guanabenz. (B) Staining intensity of p-eIF2α, normalized by intensity of eIF2α. * <i>p</i><0.05. (C) Cyto-Src activity by salubrinal and guanabenz. (D) Lyn-Src activity by salubrinal and guanabenz. Scale bars, 10 µm. <i>n</i>>7 cells.</p

Salubrinal and guanabenz inhibit cytokine-induced Cyto-Src activity.

<p>(A, B) C28/I2 cells transfected with either Cyto-Src or Lyn-Src biosensor were pretreated with TNFα or IL1β for 2 hours before incubating with salubrinal or guanabenz. (A) Effect of Salubrinal and guanabenz on cytokine-induced Cyto-Src activity. (B) Cytokine-induced Lyn-Src activity is not altered by salubrinal or guanabenz. Scale bars, 10 µm. <i>n</i>>7 cells.</p

Involvement of eIF2α in salubrinal-driven Cyto-Src activity.

<p>(A, B) C28/I2 cells were cotransfected with Cyto-Src or Lyn-Src biosensor, and eIF2α or NC siRNA, and then treated with 10 µM salubrinal for 1 hour during imaging. (A) eIF2α siRNA blocks inhibitory effect of salubrinal on Cyto-Src activity. (B) Lyn-Src activity is not altered by eIF2α siRNA. Scale bars, 10 µm. <i>n</i>>7 cells. (C) The basal level of Src activity in C28/I2 cells expressing NC or eIF2α siRNA. <i>n</i>>7 cells. * <i>p</i><0.05 compared to the corresponding NC group.</p

A proposed model of distinctive Src activities at different subcellular locations.

<p>TNFα and IL1β activate Src kinases at the cytoplasm and lipid rafts of the plasma membrane, and actin cytoskeleton and lipid rafts are essential components of the Lyn-Src activation. Salubrinal can inhibit Src kinases in the cytoplasm through phosphorylation of eIF2α, but not in the lipid rafts of the plasma membrane. In contrast, fluid flow at 5 dynes/cm² decreases integrin-mediated Src kinases in the lipid rafts of the plasma membrane, but it did not significantly affect the level of Src activation in the cytoplasm.</p