Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A) Co-staining of IRAK1 clusters and proteasome in iBMDMs. Kdo-2 Lipid A and P3C co-treatment (50 nM). Scale Bar: 10 μ . (B) Dose response of IRAK1 clustering in iBMDMs with Kdo-2 Lipid A and P3C ± 2h pre-treatment with 4uL of monensin, BD GolgiStop™ for every 3 mL of cell culture. (C-K) Kdo-2 Lipid A and P3C co-treatment (50 nM) in iBMDMs. Co-staining of IRAK1 clusters and (C) MyD88, (D) TICAM2, (E) IRAK4, (F) IRAK2, (G) TRAF6 and (H) pTBK1. Scale Bar: 10 μ . (I) Summary of IRAK1 clustering with TLR signaling components. ND: Not Determined. (J-L) IRAK1 clustering in iBMDMs 0, 2 and 3h post-treatment with 50 nM Kdo-2 Lipid A and P3C ± 30 min pre-treatment with (J) dynasore, an internalization inhibitor (20 μ M); (K) ST2825, a MyD88 dimerization inhibitor (20 μ M); and (L) thymoquinone, an IRAK1 kinase inhibitor (25 μ M). (Panels B, J-L) Data are represented as median ± MAD. (Panels J-L) Unpaired t test with Holm-Šídák’s correction. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments. See also supporting .

Article Snippet: Antibody pairs used for PLA in iBMDMs: IRAK1 Ms (SCBT sc-5288) and TRAF6 Rb (Bioss bs-1184R); IRAK1 Ms (SCBT sc-5288) and pTBK1 Rb (CST 5483S); IRAK1 Ms (SCBT sc-5288) and pTAB2 Rb (CST 8155S); IRAK2 Ms (SCBT sc-515885) and TRAF6 Rb (Bioss bs-1184R); IRAK2 Ms (SCBT sc-515885) and pTBK1 Rb (CST 5483S); IRAK2 Ms (SCBT sc-515885) and pTAB2 Rb (CST 8155S); IRAK1 Ms (SCBT sc-5288) and ASC Rb (SCBT sc-22514-R); IRAK1 Rb (CST 4504S) and NLRP3 Ms (AdipoGen AG-20B-0014-C100).

Techniques: Staining, Cell Culture

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A-K) Kdo-2 Lipid A and P3C co-treatment (50 nM each) in iBMDMs. Co-staining of IRAK1 clusters and (A) ßTrCp, (B) pellino, and (C) IRAK3. Scale Bar: 10 μ . (D) Quantification of the Pearson’s correlation coefficient (PCC) for the correlation of TRAF6 antibody staining in IRAK1 clusters. (E) Quantification of the Mander’s colocalization coefficient (MCC) for the co-occurrence of TRAF6 antibody staining in IRAK1 clusters in iBMDMs. (F) PCC and (G) MCC for pTBK1 antibody staining in IRAK1 clusters. (H-K) Proximity ligation assay in iBMDMs. (H) IRAK1-TRAF6 PLA, (I) IRAK1-pTBK1 PLA, (J) IRAK2-TRAF6 PLA, (K) IRAK1-pTBK1 PLA. (L, M) Single or co-TLR stimulation of TLR4 and TLR1/2 with Kdo-2 Lipid A and P3C (50 nM each). Quantification of (L) pp65 nuclear translocation (M) pATF2 nuclear translocation. (N) Quantification of pATF2 nuclear translocation on co-TLR stimulation of TLR4 and TLR1/2 with Kdo-2 Lipid A and P3C ± 30 min pre-treatment with dynasore, an internalization inhibitor, 20 μ M, ST2825, a MyD88 dimerization inhibitor, 20 μ M, and thymoquinone, an IRAK1 kinase inhibitor, 25 μ M. (D-G) Data are represented as mean ± SD. (H-N) Data are represented as median ± MAD. (Panels D-G) Each data point represents value from a randomly selected non-over lapping field of cells. Paired t test. (Panels H-M) Tukey’s multiple comparisons test. (Panel N) Dunnett’s multiple comparisons test. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments.

Article Snippet: Antibody pairs used for PLA in iBMDMs: IRAK1 Ms (SCBT sc-5288) and TRAF6 Rb (Bioss bs-1184R); IRAK1 Ms (SCBT sc-5288) and pTBK1 Rb (CST 5483S); IRAK1 Ms (SCBT sc-5288) and pTAB2 Rb (CST 8155S); IRAK2 Ms (SCBT sc-515885) and TRAF6 Rb (Bioss bs-1184R); IRAK2 Ms (SCBT sc-515885) and pTBK1 Rb (CST 5483S); IRAK2 Ms (SCBT sc-515885) and pTAB2 Rb (CST 8155S); IRAK1 Ms (SCBT sc-5288) and ASC Rb (SCBT sc-22514-R); IRAK1 Rb (CST 4504S) and NLRP3 Ms (AdipoGen AG-20B-0014-C100).

Techniques: Staining, Proximity Ligation Assay, Translocation Assay

Journal: The Journal of Experimental Medicine

Article Title: miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice

doi: 10.1084/jem.20101823

Figure Lengend Snippet: Exaggerated inflammatory response in miR-146a –null mice. (A and B) Serum levels of TNF (A) and IL-6 (B) in miR-146a −/− ( n = 5; KO) and WT ( n = 6) animals challenged intraperitoneally with sublethal dose of LPS (1 mg/kg). Peripheral blood was drawn at the times indicated in the graphs, and cytokine concentrations were assessed by ELISA. Results are shown as means; data are representative of two independent experiments. P-value calculations were performed using Student’s t test. (C) Kaplan-Meier survival curves of miR-146a −/− (KO) and WT mice ( n = 12 for each group) challenged with a lethal dose of LPS (35 mg/kg). Results are representative of two independent experiments. P-value calculation was performed using log-rank (Mantel-Cox) test. (D and E) Production of TNF (D) and IL-6 (E) by miR-146a −/− (KO) and WT BMDMs in response to 10 ng/ml LPS challenge. Cells were stimulated for the indicated time, and cytokine concentrations were assessed by ELISA. Data are shown as mean ± SD. Results are representative of three independent experiments. (F and G) Western blot analysis of TRAF6 and IRAK1 protein expression in WT and miR-146a −/− (KO) BMDMs (F) and CD19 + splenic B cells (G). Numbers under the blots denote relative expression normalized to β-actin expression for each sample. Two mice per genotype were analyzed. Results are representative of two independent experiments. (H and I) Quantification of Western blots in F and G. Relative TRAF6 and IRAK1 expression in WT and KO BMDMs (H) and CD19 + splenic B cells (I) is plotted. Data from two mice for each genotype were averaged and are expressed as a ratio of protein expression in miR-146a KO to WT cells. All experiments were carried in the B6/ miR-146a −/− strain of mice.

Article Snippet: Anti–mouse IRAK1 antibodies were obtained from Cell Signaling Technology, and anti-mouse TRAF6 were obtained from MBL International; anti–human IRAK1 and TRAF6 were purchased from Santa Cruz Biotechnology, Inc. Quantification of relative expression for each target protein was performed in Photoshop CS4 (Adobe), and β-actin levels were used for normalization.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: The Journal of Experimental Medicine

Article Title: miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice

doi: 10.1084/jem.20101823

Figure Lengend Snippet: Ectopic expression of miR-146a in THP-1 cells results in attenuation of inflammatory response. (A and B) Production of TNF (A) and IL-6 (B) by THP-1 stable cell lines ectopically expressing either miR-146a (THP1/146) or scramble control sequence (THP1/SCR) in response to 1 µg/ml LPS stimulation. Cells were stimulated for the indicated time, and cytokines concentrations were assessed by ELISA. Results are shown as mean ± SD. Data are representative of two independent experiments. (C) Western blot analysis of TRAF6 and IRAK1 protein expression in THP1/146 and THP1/SCR clones stimulated or not with LPS, as indicated. Numbers under the blots denote relative expression normalized to β-actin expression for each sample. Data are representative of two independent experiments.

Article Snippet: Anti–mouse IRAK1 antibodies were obtained from Cell Signaling Technology, and anti-mouse TRAF6 were obtained from MBL International; anti–human IRAK1 and TRAF6 were purchased from Santa Cruz Biotechnology, Inc. Quantification of relative expression for each target protein was performed in Photoshop CS4 (Adobe), and β-actin levels were used for normalization.

Techniques: Expressing, Stable Transfection, Control, Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Clone Assay

Journal: iScience

Article Title: ATF6 ameliorates renal warm ischemia-reperfusion injury through FHL2-mediated NF-κB signaling pathway

doi: 10.1016/j.isci.2026.115173

Figure Lengend Snippet: FHL2 interacts with TRAF6 (A) Representative IF images of FHL2 and TRAF6 expression in kidney tissues (Scale bars, 100 μm). (B–C) The expression of TRAF6 and NF-κB pathway-related proteins in HK-2 cell lines after FHL2 overexpression was examined by Western blot. GAPDH was used as a loading control. (D) CoIP experiments validate the interaction between FHL2 and TRAF6. Data represent mean ± SDs, and statistical analysis was performed using one-way ANOVA. (∗ p < 0.05).

Article Snippet: TRAF6 , Affinity Biosciences , Cat# AF5376; RRID: AB_2810280.

Techniques: Expressing, Over Expression, Western Blot, Control