Journal: BMB Reports

Article Title: Effects of α-lipoic acid on LPS-induced neuroinflammation and NLRP3 inflammasome activation through the regulation of BV-2 microglial cells activation

doi: 10.5483/BMBRep.2019.52.10.026

Figure Lengend Snippet: α-LA inhibits the expression of pro-inflammatory cytokines in LPS-treated BV-2 microglial cells. (A) Effects of α-LA on cell viability. BV-2 microglial cells were incubated with LPS (1 μg/ml) for 30 min followed by treatment with the indicated concentrations of α-LA for 24 h. Thereafter, cell viability was assessed through the MTT assay. (B and C) BV-2 microglial cells were treated with LPS (1 μg/ml) for 30 min followed by treatment with the indicated concentrations of α-LA at the suggested times. The cell-free conditioned culture medium was collected and analyzed with ELISA for TNF-α, IL-6. Data from three independent experiments are presented as means ± S.D. *≤ 0.05, **< 0.01, ***< 0.001 and are related to both LPS-induced cells and α-LA treated cells.

Article Snippet: Both TNF-α and IL-6 were quantitatively measured through an enzyme-linked immunosorbent assay (ELISA) using the mouse TNF-α and IL-6 DuoSet ELISA kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, MTT Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: IRAK4 autophosphorylation controls inflammatory signaling by activating IRAK oligomerization

doi: 10.1101/2023.12.21.572799

Figure Lengend Snippet: a) IL-2 release in WT and EL4-MyD88-GFP/IRAK4-mScarlet cells treated with DMSO or IRAK4 kinase inhibitor. IL-2 release was measured by ELISA 24 h after IL-1β stimulation. Values shown are the fold change in IL-2 release. Average values calculated from three independent experiments. Bars represent mean ± SEM. b) Treatment with the IRAK4 kinase inhibitor blocks pIRAK4 production after IL-1 stimulation. WT and EL4-MyD88-GFP/IRAK4-mScarlet cell lysates analyzed for pIRAK4 production by Western blot analysis. Cells treated with 20 µM of IRAK4 kinase inhibitor (or DMSO) for 4 h before being stimulated with 1 ng/ml of IL-1β for 30 mins in the presence of the inhibitor. c) TIRF images and kymograph analysis of DMSO control treated EL4-MyD88-GFP/IRAK4-mScarlet stimulated on IL-1 functionalized SLBs. Kymographs derived from red line overlaid TIRF images (left panel). Scale bar, 5 µm.

Article Snippet: To measure IL-2 release, we used the Mouse IL-2 DuoSet ELISA kit (R&D Systems, DY402-05) using the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Derivative Assay

Journal: bioRxiv

Article Title: IRAK4 autophosphorylation controls inflammatory signaling by activating IRAK oligomerization

doi: 10.1101/2023.12.21.572799

Figure Lengend Snippet: a) IL-2 release in EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT , IRAK4 K213/14A and IRAK4 DD . IL-2 release was measured by ELISA 24 h after IL-1β stimulation. Values shown are the fold change in IL-2 release. Average values calculated from three independent experiments. Bars represent mean ± SEM. One way Anova was used to compare the replicate means. b) TIRF images and kymograph of EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT -mScarlet stimulated on IL-1 functionalized SLBs. Kymographs derived from red line overlaid TIRF images (left panel). Scale bar, 5 µm. c) Time-series TIRF images and fluorescence-intensity time series showing the formation of a single MyD88-GFP:IRAK4 WT assembly in EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT -mScarlet. Scale bar, 1 µm.

Article Snippet: To measure IL-2 release, we used the Mouse IL-2 DuoSet ELISA kit (R&D Systems, DY402-05) using the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Fluorescence