Journal: Journal of orthopaedic surgery and research

Article Title: TRIM55 suppresses inflammatory response after spinal cord injury by accelerating the ubiquitination and degradation of TLR4.

doi: 10.1186/s13018-025-05922-w

Figure Lengend Snippet: Fig. 3 TRIM55 decreases the expression of TLR4 by promoting the K48-linked ubiquitination of TLR4. A The effect of TRIM55 on the protein level and ubiquitination level of TLR4 was evaluated using immunoblotting. B The interaction between TRIM55 and TLR4 proteins was analyzed by Co-IP. C PC12 cells were transfected with Flag-TRIM55, His-TLR4, and WT/K48R/K63R HA-UB. After IP with His, immunoblotting was performed with anti-HA. D PC12 cells were transfected with HA-UB, His-TLR4, and WT TRIM55 or MUT TRIM55. After IP with His, immunoblotting was performed with anti-HA. E PC12 cells were transfected with TRIM55 overexpression plasmids or empty vectors, and treated with CHX for 0, 6, 12, 18, and 24 h. Protein levels of TLR4 were detected using immunoblotting. F PC12 cells were transfected with TRIM55 overexpression plasmids or empty vectors, treated with MG132, and then stimulated with CHX for 0, 6, 12, 18, and 24 h. Protein levels of TLR4 were detected using immunoblotting. (n=3 independent biological replicates/group)

Article Snippet: To investigate TLR4 protein stability, transfected cells were first treated with MG132 (20 μM, MedChemExpress) for 8 h to suppress proteasomal activity.

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Transfection, Over Expression

Journal: Journal of orthopaedic surgery and research

Article Title: TRIM55 suppresses inflammatory response after spinal cord injury by accelerating the ubiquitination and degradation of TLR4.

doi: 10.1186/s13018-025-05922-w

Figure Lengend Snippet: Fig. 4 Overexpression of TLR4 abolishes the protective effects of TRIM55 on LPS-induced PC12 cells. A PC12 cells were transfected with TLR4 overexpression plasmids or empty vectors, and the expression of TLR4 was measured using qPCR. B-G After overexpressing TRIM55 and TLR4, PC12 cells were treated with LPS for 24 h. B The cell viability was detected with the CCK-8 assay. C-D The cell apoptosis was measured by TUNEL staining, and TUNEL-positive cells were counted. E-G The contents of IL-1β, IL-6, and TNF-α were measured by ELISA assay. All data are expressed as the means ± SD. (n=3 independent biological replicates/group)

Article Snippet: To investigate TLR4 protein stability, transfected cells were first treated with MG132 (20 μM, MedChemExpress) for 8 h to suppress proteasomal activity.

Techniques: Over Expression, Transfection, Expressing, CCK-8 Assay, TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: BMC Oral Health

Article Title: Edaravone attenuates doxorubicin-induced oral mucosal injury via modulation of oxidative stress, inflammatory signaling, and the SIRT1/TLR4/NF-kB/ACE2 axis in rats

doi: 10.1186/s12903-025-07148-y

Figure Lengend Snippet: Tongue tissue TLR4 and proinflammatory cytokines. A TLR4 (ng/mg protein), ( B ) TNF-α (ng/L/g), ( C ) IL-6 (ng/L/g) (mean ± SEM). DOX increased TLR4/TNF-α/IL-6; EDO reduced these levels. Statistics: TLR4/IL-6—ANOVA ± Tukey; TNF-α—Kruskal–Wallis ± Mann–Whitney. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant (adjusted p ≥ 0.05)

Article Snippet: TLR4 protein levels in tongue tissue were quantified using a rat TLR4 ELISA kit (Cusabio, Cat. No: CSB-E15822r).

Techniques: MANN-WHITNEY

Journal: Science Advances

Article Title: A neuroimmune pathway drives bacterial infection

doi: 10.1126/sciadv.adr2226

Figure Lengend Snippet: ( A ) Dose-dependent impact of PKA inhibitor H-89 on ACOD1 expression post-LPS stimulation (100 ng/ml, 6 hours). ( B ) ACOD1 expression following LPS exposure and cotreatment with dopamine (0.5 mM) and increasing doses of PKA activator forskolin (10, 50, 100, and 200 μM). ( C ) Effects of cAMP analog 8-Br-cAMP on ACOD1 expression in dopamine-cotreated cells after LPS stimulation. ( D ) CREB1 phosphorylation and ACOD1 expression in TLR4 -deficient monocytes challenged with LPS for varying durations (1 and 6 hours). ( E and F ) IP analyses revealing interactions within the TLR4 signaling complex in response to LPS and dopamine in native and DRD2 -deficient monocytes. ( G ) Phosphorylation heatmap illustrating kinase activity shifts over time (1, 3, and 6 hours) post-LPS and dopamine treatment. ( H ) Effects of MAPK1/3 inhibition (VX-11e, pluripotin, ulixertinib, all 10 μM) on CREB1 and ACOD1 regulation following LPS stimulation. ( I and J ) Influence of MAPK3 knockdown or overexpression on CREB1 phosphorylation and ACOD1 expression post-LPS challenge. ( K ) MAPK3 activation dynamics in TLR4 -knockdown THP1 cells after LPS exposure. ( L ) Protein expression profiling in DRD2 -deficient monocytes under LPS stimulation for 1 hour. All the semiquantitative data are presented as means ± SD; n = 3 biologically independent samples. Statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Surface plasmon resonance assay was used to investigate the interactions between recombinant DRD2 (rDRD2) and recombinant TLR4 (rTLR4) proteins (OriGene) using a Biacore system.

Techniques: Expressing, Phospho-proteomics, Activity Assay, Inhibition, Knockdown, Over Expression, Activation Assay