Ab7970

Venous blood samples were collected in microtubes and centrifuged (5000 rpm for 5 min 4 °C). The serum fraction was kept at −80 °C. Free T4 (fT4) was measured using the fT4 ELISA kit (DiaMetra) following the manufacturer’s instructions. T4-conjugated HRP and TMB substrate system were used for quantification.
For microscopy, thyroids (1 lobe) were embedded in paraffin. 4 µm sections were stained with hematoxylin and eosin (Sigma-Aldrich) according to the manufacturer’s instructions. For immunoperoxidase, target retrieval was performed in TEG buffer pH 9.2, as already described^{51 (link)}. Briefly, primary antibody anti-p65 (Abcam, ab7970, 1:500) was incubated overnight at 4 °C followed by incubation with the secondary anti-rabbit HRP-conjugated antibody (Dako, P0488, 1:200). Chromogenic reactions were carried out with DAB Peroxidase Substrate Kit (Vector Laboratories). For immunofluorescence analysis, thyroid sections were incubated overnight at 4 °C with anti-p65 primary antibody (Abcam, ab7970, 1:500). Sections were then incubated with secondary Alexa Fluor 488 antibody (Invitrogen); 1 μg/ml DAPI solution was used as nuclear counterstain. Zeiss Axioplan 2 microscope was used for images acquisition (20x and 63x magnification).

To determine the intracellular localization of NF-κB, immunofluorescence staining of p65 (ab7970; Abcam, Cambridge, England) in RA-FLSs was performed. In brief, RA-FLSs (1 × 10⁵ cells per well) were seeded into an eight-well chamber (Thermo Fisher Scientific) and grown to 80% confluence for 24 h. Recombinant human ORM2 (1 μg/mL; Prospec-Tany TechnoGene Ltd., Rehovot, Israel) was added to each well and incubated for 1 h. Cells were fixed with formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 3 min at room temperature. After blocking with 10% normal donkey serum at room temperature for 1 h, the cells were stained with an anti-p65 Ab (ab7970, 1:100; Abcam) and Alexa 488-conjugated donkey anti-IgG (a21206, 1:1000; Thermo Fisher Scientific). After washing with PBS three times, the nuclei were stained with DAPI (1:500; BD Biosciences, San Jose, CA, USA). The stained cells were visualized with a confocal microscope (Zeiss, LSM800, Gottingen, Germany).

Cells were harvested in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and centrifuged at 12,000×g for 15 min at 4°C. Whole cell lysate was used for SMAD7 detection. Cytosolic and nuclear fractions were prepared using standard nuclear and cytoplasmic extraction reagents (Thermo Scientific, Rockford, IL, USA). Protein concentration was measured using the Bio-Rad method. Samples (20 µg protein) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBST buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% Tween-20) for 1 h prior to incubation with a primary antibody against SMAD7 (1∶1000; ab90085; Abcam), NF-κB p65 (1∶1000; ab7970; Abcam), GAPDH (1∶2500; ab7970; Abcam) or lamin B1 (1∶1000; #13435; Cell Signaling Technology; Boston, MA, USA) at 4°C overnight, followed by incubation with an appropriate peroxidase-conjugated secondary antibody (1∶1000 dilution). Signal was visualized by chemiluminescence (Odyssey Li-COR) using GAPDH as a control. In the case of nuclear NF-κB p65, lamin B1 was employed as the loading control. Band intensity was assessed using Quantity one 4.6.2 software.