Pepsin from porcine mucosa

Cells were washed twice with phosphate buffered saline. Cell-associated collagen was solubilised by the addition of 0.5 M acetic acid (250 µl/well) and gentle agitation at 4 °C for 32 hours, before adding 0.1 mg/ml pepsin from porcine mucosa (Sigma-Aldrich) and continuing agitation for a further 16 hours. Pepsin digestion was then inhibited with 2 µg/ml pepstatin A (Sigma-Aldrich). Cell samples were stored at -80 °C until assayed. A novel quantitative immuno-blot assay for native collagen I was developed and validated by Western blot analysis of TGFβ-activated NHDF cells and supernatants (Supplementary Fig. S1). Standards (0.625–10 ng calf skin collagen I, Sigma Aldrich) and samples (100 µl) diluted with PBS (acid-soluble cell fraction, 1:10, and supernatants, 1:20), were loaded onto nitrocellulose membrane using a vacuum manifold to create protein dots. Blots were dried, blocked with 5% dried skimmed milk powder and 2% Tween 20 in PBS and incubated overnight at 4 °C with rabbit anti collagen I antibody (Abcam, Cambridge, UK) at 200 ng/ml in block buffer. The blots were then incubated with 50 ng/ml goat anti rabbit-HRP antibody (Dako, Glostrup, Denmark) for 1 hour at room temperature. Blots were visualised using chemiluminescence (Thermo-Fisher, Waltham, USA).