Fibroblast Protein Expression Analysis

Fibroblasts were lysed using 2 x Laemmli SDS sample buffer or urea buffer (8 M Urea, 1 M Thiourea, 0.5% CHAPS, 50 mM DTT, and 24 mM Spermine). Western blotting of cellular lysates was performed for β-actin (1:100.000, Sigma-Aldrich, Poole, UK), LOXL2 (1:1000, R&D Systems, Abingdon, UK), HIF1α (1:1000, BD Biosciences, Wokingham, UK), FIH (1:200, mouse monoclonal 162 C) (Wang et al., 2018 (link)), β-tubulin (1:1000, Cell Signaling Technology, London, UK), HIF1 β (1:1000, Cell Signaling Technology), p-Smad2/3 (1:1000, Cell Signaling Technology), p-ERK (1:1000, Cell Signaling Technology), active β-catenin (1:1000, Cell Signaling Technology). Immunodetected proteins were identified using the enhanced chemiluminescence system (Clarity Western Blotting ECL Substrate, Bio-Rad Laboratories Ltd, Watford, UK) or Odyssey imaging system (LI-COR), and evaluated by ImageJ 1.42q software (National Institutes of Health).