Anti smad7

Cell lysates of SFs or synovium homogenates were subjected to immunoblot with anti-Smad7 (R&D systems), anti-Flag (Sigma-Aldrich), anti-Snail (Cell Signaling), anti-Cadherin-11 (Cell Signaling), anti-acetylated-histone 3 (Cell Signaling) or anti-β-actin (Sigma-Aldrich), followed by secondary antibodies, developed with ECL Plus system (Amersham), analyzed by Biospectrum imaging system (UVP), and quantitated for the signaling intensities by ImageJ software (National Institutes of Health), as previously described9 (link)18 (link). Expression of acetylated Smad7 in TSA (Sigma-Aldrich)-treated SFs lysates was immunoprecipitated by anti-Smad7 (Santa Cruz), followed by anti-acetylated-lysine (Cell Signaling) immunoblot with small quantities (~10%) snapped for immunoblot analysis as an input control.

Expression of SMAD7, phosphorylated (p)-SMAD2, RORγT, and TGF-β were detected in both the human lung tissue homogenates samples and in the sorted CD4⁺ mouse lymphocytes using standard Western blot techniques. Isolated proteins were transferred onto a nitrocellulose membrane, and immunoblotting was performed using mouse monoclonal anti-SMAD-7 (R&D Systems, USA, dilution 1:1000), anti-p-SMAD2 (Abcam, USA, dilution 1:1000), anti-p-SMAD3 (Abcam, USA, dilution 1:1000), anti-TGF-β (Abcam, USA, dilution 1:1000), anti-ROR-γT (Abcam, USA, dilution 1:1000), anti-β-actin (Proteintech, USA, dilution 1:5000), and conjugated HRP-labeled secondary mouse or rabbit antibodies (Proteintech, USA). Images were captured using the Quantity One Analysis Software (Bio-Rad Laboratories Inc, Hercules, CA, USA).

On day 36 during the progression of arthritis, CIA mice received i.a. injections of 1 × 10⁹ VPs of LVSmad7 and LVnull into right and left ankle joints, respectively, with medium injection alone as another control group. Arthritis severity was scored on a 0 to 4 scale in each posterior paw with 0: no evidence of erythema and swelling, 1: erythema and mild swelling confined to the tarsals or ankle joint, 2: erythema and mild swelling extending from the ankle to the tarsals, 3: erythema and moderate swelling extending from the ankle to metatarsal joints, and 4: erythema and severe swelling encompassing the ankle, foot and digits, or ankylosis of the limb, as previously described18 (link)20 (link). Hematoxylin and eosin (H&E)-stained paraffin-embedded ankle joint sections were evaluated for synovial hyperplasia, cartilage erosion, and inflammatory cell infiltration, and a histologic score of 0–2 scale was assigned for each of these features (0: absent, 1: mild, 2: severe) with maximum of 6, as previously described18 (link)19 (link). Synovial immunohistochemical staining was performed with anti-Smad7 (R&D systems), anti-phosphorylated Smad2/3 (Santa Cruz), anti-IL-17 (eBioscience), anti-TNF (Santa Cruz) or isotype control IgG (Santa Cruz) with the expression intensities quantified by HistoQuest software (TissueGnostics).