The following antibodies were used: anti-PP2A (Upstate, Temecula, CA, USA), anti-phospho-ERK1/2, anti-ERK1/2, anti-Akt (Cell Signaling, Beverly, MA, USA), anti-phospho-Akt, Ser 473 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal β actin (Sigma Aldrich, St Louis, MO, USA), anti-type 1 collagen (Southern Biotech, Birmingham, AL, USA).
Recombinant human TGFβ1 was obtained from R&D Systems (Minneapolis, MN, USA). OA was purchased from Sigma Aldrich. Tissue culture reagents, Dulbecco's modified Eagle medium (DMEM) and 100× antibiotic antimycotic solution (penicillin streptomycin and amphotericin B) were obtained from Gibco BRL (Grand Island, NY, USA) and fetal bovine serum was purchased from HyClone (Logan, UT, USA). Enhanced chemiluminescence reagent and bovine serum albumin (BSA) protein assay reagent were obtained from Pierce (Rockford, IL, USA). TriReagent was purchased from the Molecular Research Center (Cincinnati, OH, USA). Primers were purchased from Operon (Huntsville, AL, USA). SMARTpool siRNA against PP2A C-subunit was purchased from Dharmacon RNA Technologies (Lafayette, CO, USA) and Hiperfect siRNA transfection reagent from Qiagen (Germantown, MD, USA).
Total RNA was isolated from dermal fibroblasts using TriReagent (Molecular Research Center) according to the manufacturer's instructions. RNA (2 μg) was reverse transcribed in a 20-μl reaction using random primers and Transcriptor First Strand synthesis kit (Roche Applied Sciences Indianapolis, IN. Quantitative (q)PCR was carried out using IQ SYBR Green mixture (Bio-Rad, Hercules, CA) on an iCycler PCR machine (Bio-Rad) using 1 μl of cDNA in triplicate with β actin as the internal control. The primers used are as follows. PP2A C-subunit α isoform: forward, 5'-GCACTTGATCGCCTACAAGA-3' and reverse, 5'-GAAATATCTTGCCCAAAGGTGT-3'. PP2A C-subunit β isoform: forward, 5'-TTCTTGTAGCATTAAAGGTGCGT-3' and reverse, 5'-CATTCCCATACTTCGCAGACA-3'.