rtlr4  (R&D Systems)

Journal: mBio

Article Title: Distinct immune properties of the N- and C-termini of the immunosuppressive domain of Ebola virus glycoprotein

doi: 10.1128/mbio.02278-25

Figure Lengend Snippet: TLR4 is involved in the capture of shed EBOV GP. ( A ) Binding and internalization assays on 293 cells stably expressing TLR4 (293-TLR4) and THP-1 cells were analyzed by western blotting. Cells were treated or mock-treated with recombinant TLR4 (rTLR4) and then cultured with medium or WT, mut 5, and mut 14 EBOV shed GP. Cells were then treated with trypsin to evaluate internalization of shed GP. Cell pellets were immunostained for TLR4, EBOV GP, and GAPDH as an internal control. ( B, C ) Induction of NFκB and NFAT. 293-TLR4 cells were transfected with NFAT-Luc ( B ) or NFκB-Luc ( C ), treated with CLI-095 or rTLR4 with or without CsA, treated or mock-treated with WT, mut 5, or mut 14 EBOV shed GP, and subjected to luciferase assays. Two-way ANOVA followed by a Tukey’s multiple comparison test: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega).

Techniques: Binding Assay, Stable Transfection, Expressing, Western Blot, Recombinant, Cell Culture, Control, Transfection, Luciferase, Comparison

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