Western blotting and analyses were performed according to previously described protocols49 (link). Human dermal fibroblasts were incubated in normal medium with or without oxymetazoline (1 μM) or isoproterenol (1 μM) for 1 hour. Cells were pretreated with 1 μM propranolol, 10 μM SB203580, 100 μM S31-201 or DMSO (vehicle control) for 30 minutes, and then stimulated with NE (10 μM) and/or sIL-6 receptor (IL-6R: 100 ng/ml). After washing with ice-cold PBS, cells were disrupted in lysis buffer (20 mM Tris-HCl pH 7.6, 140 mM NaCl, 1% Nonidet P-40) containing 1 mM phenylmethylsulfonyluoride, aprotinin (10 mg/ml) and 1 mM sodium vanadate. Lysates were centrifuged at 10,000 × g for 15 min at 4 °C and the resulting supernatants were subjected to SDS-PAGE, followed by immunoblot analysis using anti-phospho-p38 MAPK (Thr180/Tyr182) Ab, anti-p38 MAPK Ab, anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Ab, anti-44/42 MAPK (Erk1/2) Ab (Cell Signaling, Danvers, MA), anti-collagen type I Ab (SouthernBiotech, Birmingham, AL) and anti-GAPDH Ab (Santa Cruz Biotechnology, Dallas, Texas). Anti-rabbit HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used with ECL (Thermo Scientific, Rockford, IL) to image immunoblots. Quantification of protein band intensity was performed using ImageJ software version 1.46r (NIH) as previously reported49 (link).
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