Fig 1: Western blotting analysis of signalling proteins related to the TLR signalling pathway in M1 microphages. (A) Western blotting analysis of changes in the levels of the signalling proteins TRAF6, TAK1/p-TAK1, p65/p-p65, JNK/p-JNK and AP1/p-AP-1 in M1 macrophages after a-MMC treatment. (B–G) Relative density of TRAF6/ß-actin, TAK1/ß-actin, p-TAK1/ß-actin, p-JNK/JNK, p-AP-1/AP1 and p-p65/p65. The data shown are individual values with the mean ± SEM; n = 5. *P < 0.05 significantly different from the control group (0 µg/mL a-MMC); n.s. no significant difference from the control group (0 µg/mL a-MMC). One-way analysis of variance, Tukey’s multiple comparison tests.
Fig 2: Relationship between miR-1277 and TRAF6 in regulating IL-1ß-mediated CHON-001 cell viability, apoptosis, inflammation and ECM degradation. CHON-001 cells were divided into 6 groups: control, IL-1ß+miR-NC, IL-1ß+miR-1277, IL-1ß+miR-1277+pcDNA and IL-1ß+miR-1277+TRAF6. A Western blot assay was utilized for TRAF6 protein level in each group. B–E CCK-8 assay, flow cytometry analysis and EDU assay were conducted for the viability, apoptosis and proliferation of CHON-001 cells. F The protein levels of CyclinD1 and Bax in CHON-001 cells were measured via western blot assay. G ELISA kits were used for the concentrations of IL-6 and IL-8 in CHON-001 cells. H Western blot assay was employed for the protein levels of MMP13 and aggrecan in CHON-001 cells. *P < 0.05,**P < 0.01, ***P < 0.001, ****P < 0.0001
Fig 3: Circ-BRWD1 positively regulated TRAF8 expression by targeting miR-1277 in IL-1ß-treated CHON-001 cells. A, B The mRNA and protein levels of TRAF6 in IL-1ß, IL-1ß+si-NC, IL-1ß+si-circ-BRWD1, IL-1ß+si-circ-BRWD1+anti-miR-NC, or IL-1ß+si-circ-BRWD1+anti-miR-1277 treated or untreated CHON-001 cells were determined by qRT-PCR assay and western blot assay, respectively. TRAF6 mRNA expression was examined by 2-??Ct method with normalization to GAPDH. *P < 0.05, ***P < 0.001, ****P < 0.0001
Fig 4: HSP70 inhibits NF-?B-mediated inflammation through binding to TRAF6 in vitro.
Fig 5: ASB17 interacts with TRAF6. (A) HEK293T cells were co-transfected with HA-ASB17 and Flag-IRF7, Flag-STING, Flag-TBK1, Flag-IRF3, Flag-IkBa, Flag-IKKe, Flag-RIP1, Flag-TRAF6, Flag-p50, or Flag-p65 in 6-cm cell dishes for 24–36 h, respectively. A part of the cell lysates as input and remaining cell lysates were immunoprecipitated with anti-Flag antibodies. (B) HEK293T cells were co-transfected with HA-ASB17 and Flag-TRAF2, Flag-TRAF3, Flag-TRAF5, or Flag-TRAF6 in 6-cm cell dishes for 24–36 h A part of the cell lysates as input and remaining cell lysates were immunoprecipitated with anti-Flag antibodies. (C, D) HEK293T cells were co-transfected with HA-TRAF6 and Flag-ASB17 in 10-cm cell dishes for 24–36 h A part of the cell lysates as input and remaining cell lysates were immunoprecipitated with anti-HA antibody (C) or anti-Flag antibody (D). (E) HEK293T cells were co-transfected with Flag-TRAF6 and HA-ASB17 in 6-cm cell dishes for 24–36 h. A part of the cell lysates as input and remaining cell lysates were immunoprecipitated with IgG or anti-HA antibodies. (F) A part of THP-1 stably expressing ASB17 cell lysates as input and remaining cell lysates were immunoprecipitated with IgG or anti-Flag antibodies. (G) THP-1 stably expressing ASB17 cells were stimulated with LPS (1 µg/ml) in different timepoints (0 and 2 h) in 10-cm cell dishes. A part of the cell lysates as input and remaining cell lysates were immunoprecipitated with IgG or anti-Flag antibodies. (H) HEK293T cells were co-transfected with plasmids as indicated. Subcellular localizations of HA-ASB17 (green), Flag-TRAF6 (red), and nucleus marker DAPI (blue) were analyzed under confocal microscopy. Scale bar, 20 µm. (I) HEK293T cells were co-transfected with plasmids as indicated, biomolecular fluorescence complementation (BiFC) assays for detection of interactions between ASB17 and TRAF6. All bands were immunoblotted with the indicated antibodies.
Supplier Page from Abcam for Anti-TRAF6 antibody