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. 2011 Jun 30;6(6):e21484. doi: 10.1371/journal.pone.0021484

Figure 4. Desensitization of PAR-1 with Thrombin (2.5 U/ml) reduces GrK-induced IL-6, IL-8 and MCP-1 production in lung fibroblasts.

Figure 4

(A–C) HFL were treated with thrombin (2.5 U/ml) for 10 min, monolayers were then rinsed three times with fresh media and incubated with GrK (200 nM) for 24 h. Supernatants were collected and IL-6, IL-8 and MCP-1 levels were analyzed by ELISA. Supernatants were normalized using cell counts. Data are expressed as % change over media control ± SEM from three separate experiments run in triplicate. (D) GrK (200 nM) and thrombin (2.5 U/ml) induced ERK1/2 phosphorylation following 10 min incubation. Pre-treatment (PT) of HFL with thrombin (2.5 U/ml) for 10 min prior to treatment with GrK (200 nM) for an additional 10 min decreased ERK1/2 phosphorylation when compared to monolayers incubated with GrK (200 nM) alone. ERK1/2 phosphorylation was assessed by Western blotting of cell lysates using anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies. Protein loading was verified and normalized using β-tubulin. Data represent the mean ± SEM of at least three independent experiments. + p<0.05 when compared to media control (1% DMSO in DMEM); ** p<0.05 compared to GrK (200 nM) treated cells.