A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System

Figure 5

Effect of PMA-mediated ROS generation on tyrosine kinase activation. (a) HT-29 cells in 6-well plate were incubated in HBSS or Opti-MEM and treated with PMA (200 nM) for the indicated times. Shown are immunoblots of pY397 FAK and total FAK. (b) Fold change of pY397 FAK was calculated (, ±SD). (c–e) HT-29 cells were pretreated with NAC (10 mM) or DPI (10 μM) for 1 h and then stimulated with PMA (200 nM) for 30 min. Cells were grown in 10% FBS DMEM. (c) Shown are immunoblots of pY397 FAK, pY402 Pyk2, pY416 Src, and GAPDH as the loading control. (d) Fold change of pY397 FAK, pY402 Pyk2, and pY416 Src was calculated (, ±SD). (e) Shown are immunostainings for pY397 FAK (red), actin stress fibers (green), and merge (red and green). Scale bar, 20 μm (). vs. control or vs. PMA.

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