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2 protocols using rabbit anti traf6

1

Immunofluorescence Staining of TRAF6 and NF-κB

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All the slides containing cells were fixed in 4% paraformaldehyde for 15 min, permeated in 0.1% Triton X-100 for 5 min, and then blocked in 5% goat serum for 1 h. HASMCs were stained or costained with rabbit anti-TRAF6 (Cat#ab62488, Abcam; 1:50) and mouse anti-NF-κB (Cat#sc8008, Santa Cruz; 1:50) overnight at 4°C. The cells were incubated with secondary goat anti-rabbit IgG-PE conjugate (Cat#sc3739, Santa Cruz; 1:100) or goat anti-mouse IgG-FITC conjugate (Cat#sc2010, Santa Cruz; 1:100). The slides were washed 3 times in between each step, and lastly, incubated with anti-fade reagent with DAPI (Cat#8961, CST, USA). The cells were observed under a confocal laser scanning microscopy (LSM710, ZEISS, Germany). We took 4 images for every slide randomly, and repeated all the experiments for at least 3 times.
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2

Western Blot Analysis of Bone Cells

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Proteins from total BMCs and from BMSCs were extracted in cell lysis buffer (Cell Signaling, EuroClone) after 2 days of culture, and the concentration was determined by the BCA protein assay reagent (Pierce, EuroClone). Western blotting was performed as previously described (Sabbieti et al. 2010) (link). Membranes were immunoblotted in blocking buffer with specific antibodies: rabbit anti-TNFα and rabbit anti-NF-κB (BioLegend, Microtech SrL, Napoli, Italy) both diluted 1:500; mouse anti-RANKL, rabbit anti-TRAF6, rabbit anti-CXCL12 and rabbit anti-TGFβ (Abcam, Prodotti Gianni) all diluted 1:600; rabbit anti-PPRγ (Santa Cruz Biotechnology, Inc. DBA) diluted 1:300 and rabbit anti-Osterix (Santa Cruz Biotechnology, DBA) diluted 1:300. After washing with PBS-T, blots were incubated with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG or with HRP-conjugated rabbit anti-mouse IgG (Cell Signaling, EuroClone) both diluted 1:50,000. Immunoreactive bands were visualized using LiteAblot Turbo luminol reagents (EuroClone) and Hyperfilm-ECL film (EuroClone) according to the manufacturer's instructions. To normalize the bands, filters were stripped and re-probed with a monoclonal anti-α-tubulin (Sigma-Aldrich). Band density was quantified densitometrically.
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