Hspd1

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).

For analysis of protein levels by Western blot, whole tissue lysates were used from either the proteomic analysis or were prepared as described previously (34 (link)). Proteins were separated on precast SDS-PAGE 4–12% criterion gels (Bio-Rad) and transferred to polyvinylidene difluoride or nitrocellulose membranes. Site-specific antibodies directed to acetylated α-tubulin (Sigma, T7451), HSPA1 (Enzo Life Sciences, ADI-SPA-810), HSPA2 (Proteintech group, 66291-1), HSPB1 (Enzo Life Sciences, ADI-SPA-800), HSPB5 (Enzo Life Sciences, ADI-SPA-223), HSPB7 (abcam, ab150390), HSPD1 (Enzo Life Sciences, ADI-SPA-805), HSPA4 (Cell Signaling, 3303S), HSP90 (Cell Signaling, 4874S), α-tubulin (Sigma, T9026), tyrosinated tubulin (Sigma, T9028), detyrosinated tubulin (abcam, ab48389), and GAPDH (Cell Signaling, 2118S; Fitzgerald, 10R-G109a) were used to detect the proteins which were visualized with an enhanced chemiluminescence detection kit (Amersham) and scanned with Amersham Imager 600. Protein levels were determined by densitometric analysis and normalized to GAPDH.