human il23  (R&D Systems)

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-23p19 and IL-27/IL-35Ebi3 can form a heterodimer, termed IL-39. (A) Detection and characterization of human IL-39 by immunoprecipitation (IP) and Western blot (WB) analyses under reducing conditions. (B) Schematic of the cDNA constructs used to genetically engineer mouse p19 and Ebi3 proteins. hPGK: human phosphoglycerate kinase 1 promoter. (C) Detection and characterization of mouse IL-39 recombinant proteins by IP and Western blot analyses under reducing conditions. (D) Coomassie Blue gel of the recombinant mouse IL-39 characterized on nonreducing polyacrylamide gels. Arrow indicates recombinant IL-39. (A, C, and D) Blots are representative of three independent experiments.

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Immunoprecipitation, Western Blot, Construct, Recombinant

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: In vitro activated B cells expressed IL-39. (A, B) Primary B cells were sorted from 8-weeks-old female C57BL/6 mice by B220 microbeads, stimulated for 48 h with LPS, and analyzed by FACS. (A) The percentages of IL-39-expressing B cells and (B) statistical analysis of the percentage of IL-12 family cytokine subunits are shown. Isotype was used as the staining control. (C) The concentration of IL-39 in the cultured supernatant from LPS-stimulated B cells was measured by sandwich ELISA by using anti-p19 and Ebi3 antibody as coated and detected antibody, respectively. (B and C) Data are shown as mean + SEM (n = 8) from one experiment representative of three other similar experiments. (D) Detection of mouse natural IL-39 proteins in the cultured supernatant on days 3 after B cells were stimulated with LPS by IP and Western blot analyses under reducing conditions. Blots are representative of three independent experiments *p < 0.05, **p < 0.01, ***p < 0.001 (two tailed Student’s t-test).

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: In Vitro, Expressing, Staining, Control, Concentration Assay, Cell Culture, Sandwich ELISA, Western Blot, Two Tailed Test

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-39 induces inflammation in lupus-like Mice. (A) GL7+ B cells were sorted from 8-month-old female lupus-like MRL/lpr mice were sorted by FACS and infected with control, IL-39 subunits p19 or Ebi3-specific shRNA. On day 1 after infection, p19 and Ebi3 mRNA expression were analyzed by qPCR. (B–D) A 5 × 106 control, p19 or Ebi3-specific shRNA-infected GL7+ B220+ B cells per mouse were i.v. injected into 8-week-old female lupus-like MRL/lpr mice (six mice per group). Eight-week-old female lupus-like MRL/lpr mice into which either no cells, or transferred with control shRNA-infected GL7+ B cells, were used as no-cells transfer (None) and shRNA (Control) controls respectively. (B) On day 14 after cell transfer, spleens were harvested and photographed (representative of six spleens per experiments from three independent experiments). (C) Proteinuria was measured on day 0 and day 4 after cell transfer. (D) Absolute numbers of B220+ B and GL7+ B per spleen on days 7 after cell transfer. (A, C, and D) Data are shown as mean + SEM (n = 6) from one experiment representative of two other similar experiments. *p < 0.05, **p < 0.01 (two tailed Student’s t-test).

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Infection, Control, shRNA, Expressing, Injection, Two Tailed Test

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-39 signals through IL-23R and gp130 receptor subunits and activates STAT1 and STAT3 pathways. (A) Primary B cells were sorted from 8-week-old female C57BL/6 mice by B220 microbeads, stimulated for 48 h with LPS, washed, and starved for 2 h in serum- free medium (0.5% BSA), followed by stimulation for 30 min with medium, p19, Ebi3, or IL-39. STAT activation was analyzed by Western blotting. Blots are representative of four independent experiments. (B) Primary B cells were sorted from 8-week-old female C57BL/6 mice by B220 microbeads, stimulated for 24 h with LPS in the presence of medium p19, Ebi3, or IL-39. IL-39 subunits (p19 and Ebi3) mRNA was determined using qPCR assay. Data are shown as mean + SEM (n = 8) from one experiment representative of two other similar experiments. (C) Control or IL-23R, IL-27Ra, gp130-specific shRNA-infected B cells described in Supporting Information Fig. 3C were stimulated for 2 days with LPS, washed, and starved for 2 h in serum-free medium (0.5% BSA), followed by stimulation for 30 min with IL-39. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr701-STAT1 (pSTAT1) and total STAT1 (upper and left panel) or phosphor-Tyr705-STAT3(pSTAT3)andtotalSTAT3(upper and right panel). Band intensities of pSTAT1 and STAT1 or pSTAT3 and STAT3 were quantified by ImageProPlus 5.0 software. The density ratios of phosphorylated to total protein compared to control shRNA-infected group are shown as mean ± SEM (n = 3) of three independent experiments (lower panel). *p < 0.05, **p < 0.01 (two tailed Student’s t-test).

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Activation Assay, Western Blot, Control, shRNA, Infection, SDS Page, Software, Two Tailed Test