Quantifying Protein Abundance via Western Blot and IHC

The relative abundance of several proteins was analyzed using Western blot or immunohistochemistry (IHC) as previously described [5 (link), 13 (link)]. Primary antibodies for HSPD1 (1 : 100; Enzo Life Sciences), E-cadherin (1 : 100; Abcam), Vimentin (1 : 100; Abcam), α-SMA (1 : 75; Sigma-Aldrich), 3-NT (1 : 100; Abcam), Trx (1 : 100; Abcam), VCAM-1 (1 : 10;0 Abcam), CD3 (1 : 50; Abcam), and Bax (1 : 100; Cell Signaling Tech) were applied according to the manufacturer's instructions. Secondary antibodies (1 : 1000; Jackson ImmunoResearch) were also applied according to the manufacturer's instructions. All immunohistochemistry staining and collagen staining were analyzed as the following steps: under a microscope (400x), we move the slices randomly and take 20–25 pictures per slice, then analyze by Image Pro-Plus 6.0 Software (Media Cybernetics, USA) to quantify the abundance of our target protein/gene. Parameter of IOD (integrated optical density) was employed to do this work. To prove our assumption with a second method, we also quantified tyrosine-nitrosylated proteins by fluorescence immunoblotting of 3-NT (Abcam; 1 : 100). Secondary antibodies were species-specific FITC-conjugated IgG (1 : 500, Invitrogen).