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.
Rabbit anti traf6
Manufactured by Abcam
Sourced in Italy
Rabbit anti-TRAF6 is a primary antibody that recognizes the TRAF6 protein. TRAF6 is an E3 ubiquitin-protein ligase that plays a critical role in the activation of NF-kappa-B and MAPK signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Goat anti mouse igg fitc conjugate, by Santa Cruz Biotechnology (1 mentions)
Mouse anti nf κb, by Santa Cruz Biotechnology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Liteablot turbo luminol reagents, by Euroclone (1 mentions)
Hyperfilm ecl film, by Euroclone (1 mentions)
Rabbit anti osterix, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti tgf β, by Abcam (1 mentions)
Rabbit anti cxcl12, by Abcam (1 mentions)
Mouse anti rankl, by Abcam (1 mentions)
Hrp conjugated rabbit anti mouse igg, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
2 protocols using rabbit anti traf6
1
Immunofluorescence Staining of TRAF6 and NF-κB
2
Western Blot Analysis of Bone Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!