RNA was isolated using TriFast reagent (Peqlab, Erlangen, Germany) according to the manufacturer's instructions up to the point of phase separation. The aqueous phase was transferred to an RNeasy column (Qiagen, Hilden, Germany) and processed according to the manufacturer's protocol, RNA quality was measured with the Nanodrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Quantitative real-time PCR (qRT-PCR) was performed using the StepOne Plus system (Applied Biosystems, Darmstadt, Germany) and the PowerSYBR green RNA-to-CT TM kit (Applied Biosystems). Primers were designed with the Primer3 software
28 and were designed to span exon boundaries in order to avoid amplification of contaminating genomic DNA (see
Supplementary Table S4). Human
GAPDH,
29 mouse
Gapdh30 as well as human/mouse
18s rRNA29 primer sequences for internal standard have been reported previously. Using 30ng of RNA per reaction, PCR cycling conditions for reverse transcription one-step PCR were as recommended by the manufacturer (StepOne SoftwareTM v2.1; Applied Biosystems). The relative RNA expression level was determined from triplicates of three independent assays by the ΔΔCt method and expressed as 2ΔΔCt for fold change. Statistical analysis was performed using Student's
t-test (Fold changes >2 were considered as statistical significant). Western blot analysis was conducted as described previously.
30 Briefly, whole protein lysates from human and mouse tissues were extracted in ice-cold lysis buffer (50 mMTris–HCl [pH 8.0], 150 mM NaCl, 1% [v/v] NP-40, 1 g/L SDS, 1 g/L Na-Desoxycholate) with protease inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO, USA) on ice for 10 minutes centrifuged at 600
g for 20 minutes and then supernatants were harvested and stored at −20°C. Protein quantification was performed according to a standard method (BCA Protein Assay Reagent, Thermo Scientific). For each gel lane, 40 μg protein was denatured in 5 x Laemmli buffer at 95°C for 5 minutes. Disulfide bonds were reduced by adding 1M Dithiothreitol (DTT, Sigma-Aldrich Corp.). Proteins were loaded on 10% SDS–PAGE, transferred onto nitrocellulose (Hybond C, GE Healthcare, Freiburg, Germany) and incubated with the polyclonal goat anti-CCP5 antibody (human tissue: T-20; Santa Cruz Biotechnology, Inc., Heidelberg, Germany; 1:300 dilution, mouse tissue: G-19, Santa Cruz Biotechnology, Inc.; 1:250 dilution). Using mouse anti-goat secondary antibody (1:10,000 dilution; Thermo Scientific) as a conjugate, detection was carried out using ECL plus (Thermo Scientific) and the Fusion SL imaging system (Peqlab). Blots were stripped using Western blot stripping solution (Thermo Scientific) and re-probed with rabbit anti-GAPDH (1:1000 [human] and 1:500 [mouse] dilution, ab9485; Abcam, Cambridge, UK) as a loading control.