Phospho smad3

To examine the changes in renal morphology, we stained formalin‐fixed, paraffin‐embedded sections (3 µm) with haematoxylin and eosin or a Masson's trichrome staining kit (ScyTek Laboratories, West Logan, UT) according to the manufacturer's instructions and as previously described.29, 30 Immunohistochemistry was performed in paraffin sections using a microwave‐based antigen retrieval method.31 The primary antibodies used in this study included antibodies against TNF‐α, IL‐1β, TGF‐β1, fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA), F4/80 (AbD Serotec, Kidlington, UK), phospho‐Smad3 (Rockland Immuno‐chemicals, Gilbertsville, PA), α‐SMA (Sigma, St. Louis, MO), collagen I (Southern Biotech, Birmingham, AL) and phospho‐nuclear factor κ light chain enhancer of activated B cells (NF‐κB)/p65 (Abcam, Cambridge, MA). Positive signals were analysed with the quantitative Image Analysis System (Image‐Pro Plus 7.0; Media Cybernetics, Bethesda, MD) as described previously.32, 33

The pathological changes in synovial tissues and joints were examined in paraffin-embedded tissue sections (4 μm)by hematoxylin-eosin (HE) staining. Immunohistochemistry (IHC) was performed on paraffin sections using the microwave-based antigen retrieval technique. The antibodies used in this study were as followed: CD3, IL-17, IL-6 and Foxp3 (Abcam, Cambridge, United Kingdom), TNF-α, IL-1β, TGF-β, TGF-β receptor II, Smad7 (Santa Cruz, California, United States), F4/80 (Serota, Raleigh, North Carolina, United States), MCP-1 (eBiosience, San Diego, CA, United States),phospho-Smad3 (Rockland, Philadelphia, United States), phospho-p65 (Cell signaling, Beverly, MA, United States), rabbit anti-rat secondary antibody, rabbit anti-goat secondary antibody and anti-rabbit polymer (DAKO, Carpinteria, CA, United States). Expression levels of TGF-β,TGF-β receptor II, Smad7, TNF-α, IL-1β, MCP-1, IL-17, and IL-6 in synovial tissues were analyzed and determined using the quantitative Image Analysis System (AxioVision 4, Carl Zeiss, Germany) as previously described (1 (link)). The number of cells positive for phopsho-p65, phospho-Smad3, T-bet, Gata3, CD3, andF4/80 were counted in 5 consecutive high power fields (40x) by means of a 0.0625-mm² graticule fitted in the eyepiece of the microscope and expressed as cells per millimeters squared.

Paraffin sections were evaluated by Immunohistochemistry using a microwave-based antigen retrieval method 21 (link). The primary antibodies used in this study included antibodies against TNF-α, IL-1β, TGF-β1, fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA), F4/80 (AbD Serotec, Kidlington, UK), phospho-Smad3 (Rockland, Immuno-chemicals, Gilbertsville, PA), α-SMA (Sigma, St. Louis, MO), collagen I (Southern Biotech, Birmingham, AL), and phospho-NF-κB/p65 (Abcam, Cambridge, MA, USA). Positive signals were analysed with the quantitative Image Analysis System (Image-Pro Plus 7.0, Media Cybernetics, Bethesda, MD, USA) as described previously 6 (link), 22 (link).