Journal: The Journal of Clinical Investigation
Article Title: α -Ketoglutarate protects against cartilage damage via epigenetically driven metabolic reprogramming in osteoarthritis models
doi: 10.1172/JCI172380
Figure Lengend Snippet: ( A ) IB analysis of K63-linked ubiquitination of TRAF6 in HEK293T cells transfected to express HA-TRAF6 with or without Flag-tagged K63-linked ubiquitin (Flag-K63-Ub) and MYC-tagged UBE2O (MYC-UBE2O). ( B ) Densitometry of the bands in A , showing the ubiquitination (Ub) of TRAF6, presented relative to results obtained with cells transfected to express empty vector, set as 100%. ( C ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 in IL-1β–stimulated chondrocytes in the presence of BPTES. Blots are representative of 3 independent experiments. ( D ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 that was immunoprecipitated and TRAF6 from IL-1β– and DM-αKG–stimulated chondrocytes. ( E ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 immunoprecipitated from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG. ( F ) Protein quantitative analysis of the nuclear p65 from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG for 60 minutes. ( G and H ) Quantitative analysis of qRT-PCR ( G ) and Western blot ( H ) analysis for the indicated anabolic and catabolic factors from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG. ( I ) IHC staining for pIκBα, p-p65, and quantification of pIκBα- and p-p65–positive cells at 25 weeks in mice fed a standard diet (SD) or HFD. ( J ) IHC staining for pIκBα, p-p65, and quantification of pIκBα- and p-p65–positive cells in Gls1 −/− mice and their Gls1 fl/fl control littermates after DMM surgery. veh, vehicle. ( K ) Schematic depicting the described findings: αKG inhibited the TRAF6 ubiquitination via inducing the expression of UBE2O in OA chondrocytes. The data are presented as box plots or as the mean ± SEM, and the dots represent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.
Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against SOX9 (Abcam, ab3697; 1:1,000); Collagen II (Abcam, ab85266; 1:1,000); MMP3 (Abcam, ab52915; 1:1,000); MMP13 (Proteintech, 18165-1-AP; 1:1,000); ADAMTS5 (Abcam, ab41037; 1:200); NOS2 (Abcam, ab178945; 1:1,000); SLC1A5 (Sigma, HPA035239; 1:1,000); GLS1 (Proteintech; 12855-1-ap; 1:1,000); GS (Abcam, ab176562; 1:1,000); NF-κB p65 (Santa Cruz Biotechnology; sc-8008; 1:1,000); lamin A/C (Santa Cruz Biotechnology; sc-6215; 1:5,000); phospho-IKKα/β (Cell Signaling Technology; 2694P; 1:1,000); IKKα/β (Abcam, ab178870; 1:1,000); phospho-IκBα (Affinity, AF2002; 1:1,000); IκBα (Affinity, AF5002; 1:1,000); TRAF6 (Santa Cruz Biotechnology; sc-8409; 1:200); K63 Ub (Cell Signaling Technology; 5621S; 1:1,000); and GAPDH (Abcam, ab8245; 1:2,000).
Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Immunohistochemistry, Control, Expressing
Bioss is a verified supplier 
Bioss manufactures this product 
