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Figure 5 | Cell Communication and Signaling

Figure 5

From: NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes

Figure 5

Involvement of PKC-α in BK-induced ROS generation and MMP-9 expression in RBA-1. (A) Cells were pretreated without or with GF109203X (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. (B) Cells were pretreated with or without GF109203X (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. (C) Cells were pretreated with or without BAPTA (BAP, 30 μM) or Gö (1 μM) for 1 h and then treated with 10 nM BK for the indicated time intervals (upper part) or 5 min (lower part). The membrane and cytosol fractions were prepared and analyzed by Western blotting. (D) Cells were pretreated without or with GF or Gö for 1 h before exposure to 10 nM BK for 5 min. The Nox activity and ROS generation were analyzed. (E) Cells were transfected with scramble (scra) or PKC-α siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. (F) Cells were pretreated without or with Gö (1 μM), BAPTA (30 μM), or TG (1 μM) for 1 h before exposure to 10 nM BK for 5 min. The cell lysates were collected and analyzed by Western blotting using an anti-phospho-serine (p-Ser), p47phox, or GAPDH antibody. Data are expressed as the mean ± SEM (n = 3). *P < 0.05; #P < 0.01, as compared with the respective values of cells stimulated with vehicle (C) and BK (A, B, D-F) only. The figure represents one of three similar experiments.

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