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. 2014 Feb;50(2):301–315. doi: 10.1165/rcmb.2012-0522OC

Figure 1.

Figure 1.

Effect of p38 mitogen-activated protein kinase (MAPK) inhibition on glucocorticoid receptor (GR) site-specific phosphorylation. (A) Top: Cells were pretreated with SB203580 (10 μM) for 1 hour before fluticasone propionate (FP) was added for an additional 2 hours. Cells were lysed, and whole cell lysate extracts were prepared and assayed for serine (S)203, S211, S226, GR, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by immunoblot analysis. Results are representative of three separate blots. (B) Scanning densitometry of three representative immunoblots with each condition normalized over the area density of the corresponding GAPDH content. The results are expressed as the fold increase over basal values. *P < 0.05 compared with untreated cells; **P < 0.01 compared with untreated cells; ***P < 0.001 compared with untreated cells; #P < 0.05 compared with untreated cells; &P < 0.05 compared with fluticasone-treated cells. NS = not significant. (C) Cells were pretreated with SB203580 (10 μM) for 1 hour before TNF-α (10 ng/ml) was added for an additional 15 minutes. Cells were lysed, and whole cell lysate extracts were prepared and assayed for phospho-ATF2 (pATF2) or total ATF2 and GAPDH by immunoblot analysis. Results are representative of three separate blots. (D) Cells were transfected for 48 hours with 100 nM p38 MAPK small interfering RNA (siRNA) or control siRNA before FP was added for an additional 2 hours. Cells were lysed, and whole cell lysate extracts were prepared and assayed for S203, S211, S226, p38, GR, and GAPDH by immunoblot analysis. Results are representative of three separate blots. (E) Scanning densitometry of three representative immunoblots with each condition normalized over the area density of the corresponding GAPDH content. *P < 0.05 compared with untreated cells transfected with control siRNA; #P < 0.05 compared with untreated cells transfected with control siRNA; &P < 0.05 compared with FP-treated cells transfected with control siRNA.