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

Journal: Oncoimmunology

Article Title: Systemic administration of β-glucan of 200 kDa modulates melanoma microenvironment and suppresses metastatic cancer.

doi: 10.1080/2162402X.2017.1387347

Figure Lengend Snippet: Figure 3. BG34-200 treatment augments systemic activation of T cells recognizing melanoma antigen. (A) Splenocytes or TDLN cells were stimulated with gp10025-33 peptide. IFN-g and IL-2 levels in the culture media were assessed using ELISA. B. Frequencies of CD3, CD4, CD8 T cells and their expression of CD62L and CD44 in spleen and TDLN were determined by FACS analysis. C. TDLN cells were subjected to intracellular cytokine staining for determining the frequency of gp10025-33 -specific T cells. (A) and (B) were graphed as means ± SD. Each data point represents one of three replicates of samples from individual TDLN or spleen. * p < 0.05, ** p < 0.01. Significance was determined using Student’s t test.

Article Snippet: Mouse IFN- and IL-2 ELISA kits were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: BMC Biotechnology

Article Title: Extracellular overproduction of E7 oncoprotein of Iranian human papillomavirus type 16 by genetically engineered Lactococcus lactis

doi: 10.1186/s12896-019-0499-5

Figure Lengend Snippet: Panel a : The levels of E7-specific IgG and SIgA antibodies in serum (left) and vaginal discharges (right), respectively, on day 41; the sera and vaginal discharge samples were tested by indirect ELISA using recombinant human papillomavirus type 16 protein E7 as the coating antigen. Goat-anti-mouse IgG-H&L (HRP) antibody and Goat Anti-Mouse IgA alpha chain (HRP) were used as a secondary antibody. The absorbance of each microwell was read on a spectrophotometer at 450 nm. Error bars represent the mean ± standard error value of each group ( P < 0.001). Panels b and c : Detection of E7-specific T-lymphocytes using an IL-2 (panel b ) and IFN-γ (panel c ) ELISPOT assay in the splenocytes (left) and intestinal mucosal lymphocytes (right); female C57BL/6 mice were immunized orally with 10 9 CFU of Lactococcus lactis containing rE7 on days 1, 2, 3, 14, 15, 16, 29, 30, and 31. In order to measure cytokine production from the cells, on day 41 (ten days after the last immunization), intestinal mucosal lymphocytes and splenocytes were collected and stimulated with MHC class I - restricted peptides and MHC class II - restricted peptides (HPV-16 E7 49–57 and HPV-16 E7 30–67 , respectively). Then, the level of E7-specific IFN-γ-producing CD8 + and CD4 + T cells and E7-specific IL-2-producing CD8 + and CD4 + T cells was calculated via mouse IFN-γ and IL-2 ELISPOT kit, respectively. The diagram demonstrates the number of spots per 10 4 splenocytes and intestinal mucosal lymphocytes after stimulation. The results are presented as mean ± SD (n 10). PBS (Control group); LLEV: Induced L. lactis harboring pNZ8123; LLE7BF: Induced L. lactis harboring pNZ8123-HPV16-E7 produced through batch fermentation; LLE7FBF: Induced L. lactis harboring pNZ8123-HPV16-E7 produced through fed-batch fermentation; LLOE7BF: Induced L. lactis harboring pNZ8123-HPV16-optiE7 produced through batch fermentation; and LLOE7FBF: Induced L. lactis harboring pNZ8123-HPV16-optiE7 produced through fed-batch fermentation

Article Snippet: Afterwards, the number of E7-specific IFN-γ-producing CD8 + and CD4 + T cells and E7-specific IL-2-producing CD8 + and CD4 + T cells were measured using mouse IFN-γ ELISPOT kit and mouse IL-2 mouse ELISPOT kit, respectively, according to the manufacturer’s instructions (R&D Systems, Inc., Minneapolis, MN).

Techniques: Indirect ELISA, Recombinant, Spectrophotometry, Enzyme-linked Immunospot, Control, Produced