Journal: Molecules

Article Title: Expression, Purification, and Characterization of Interleukin-11 Orthologues

doi: 10.3390/molecules21121632

Figure Lengend Snippet: Crystal structure of human IL-11 (PDB entry 4MHL ). α-Helices A to D are labelled. Dashed lines mark the non-resolved regions. The residues comprising the proposed sites for IL-11 interaction with IL-11Rα (site I, green) and gp130 (site II, yellow; site III, grey) receptors are indicated. The residues not conserved in either macaque or mouse IL-11 are shown in cyan (see ).

Article Snippet: The deubiquitinating protease Usp2 was prepared as described in [ ]. sIL-11Rα was from Sino Biological, Inc. (#10252-H08H, Beijing, China). pHUE and pHUsp2-cc vectors were kindly provided by Dr. Rohan T. Baker [ ].

Techniques:

Journal: Molecules

Article Title: Expression, Purification, and Characterization of Interleukin-11 Orthologues

doi: 10.3390/molecules21121632

Figure Lengend Snippet: Kinetics of the interaction of macaque/murine rIL-11 with sIL-11Rα (panels A and B , respectively) at 25 °C, monitored by SPR spectroscopy (PBS, 0.05% TWEEN 20, pH 7.4 buffer). The biotinylated rIL-11 was immobilized on the surface of NLC sensor chip. Concentrations of sIL-11Rα used as an analyte are indicated above the curves. Grey curves are experimental, while black curves are theoretical, calculated according to the heterogeneous ligand model (1) (see for the fitting parameters).

Article Snippet: The deubiquitinating protease Usp2 was prepared as described in [ ]. sIL-11Rα was from Sino Biological, Inc. (#10252-H08H, Beijing, China). pHUE and pHUsp2-cc vectors were kindly provided by Dr. Rohan T. Baker [ ].

Techniques: Spectroscopy

Journal: Molecules

Article Title: Expression, Purification, and Characterization of Interleukin-11 Orthologues

doi: 10.3390/molecules21121632

Figure Lengend Snippet: Kinetic and equilibrium dissociation constants describing the SPR data ( Figure 5 ) on interaction of macaque/mouse rIL-11 with sIL-11Rα according to the heterogeneous ligand model (1). Standard deviations estimated from the global fit of three kinetic SPR curves measured at different analyte concentrations are indicated.

Article Snippet: The deubiquitinating protease Usp2 was prepared as described in [ ]. sIL-11Rα was from Sino Biological, Inc. (#10252-H08H, Beijing, China). pHUE and pHUsp2-cc vectors were kindly provided by Dr. Rohan T. Baker [ ].

Techniques:

Journal: Molecules

Article Title: Expression, Purification, and Characterization of Interleukin-11 Orthologues

doi: 10.3390/molecules21121632

Figure Lengend Snippet: IL-11-induced STAT3 activation in a HEK-Blue™ IL-6 cell line transfected with human IL-11Rα gene, monitored by STAT3-induced secretion of SEAP (QUANTI-Blue™ assay) for murine, macaque and human rIL-11. Standard deviations estimated from the duplicate measurements are shown. The dashed curves are theoretical fits to the experimental curves using Boltzmann function.

Article Snippet: The deubiquitinating protease Usp2 was prepared as described in [ ]. sIL-11Rα was from Sino Biological, Inc. (#10252-H08H, Beijing, China). pHUE and pHUsp2-cc vectors were kindly provided by Dr. Rohan T. Baker [ ].

Techniques: Activation Assay, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130

doi: 10.3390/ijms25031363

Figure Lengend Snippet: sgp130Fc inhibits IL6, IL11, OSM, and CT1-induced STAT3 activation. ( A ) ELISA of IL6R, IL11RA, LIFR, and OSMR in the conditioned supernatant of A549, primary human hepatocytes (HEP), and primary human hepatic stellate cells (HSC) 17 h after plating. Data are shown as box-and-whisker plots with median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers). Western blots of p -STAT3 and STAT3 in CNTF, CT1, IL6, IL11, LIF (5 ng/mL)-stimulated ( B ) A549, ( C ) HEP, ( D ) HSC (15 min) and ( B – D ) immunoblots of p -STAT3, STAT3, GAPDH in OSM (5 ng/mL)-stimulated A549, HEP, HSC (15 min) in the presence or absence of sgp130Fc (5 µg/mL). ( A – D ) n = 4 biological replicates. BL: baseline. n.d. not detected.

Article Snippet: The levels of IL6R, IL11RA, LIFR, and OSMR in an equal volume of cell culture supernatant were quantified using Human IL-6R alpha Quantikine ELISA kit (DR600, R&D Systems, Minneapolis, MN, USA), Human interleukin 11 receptor alpha (IL11RA) ELISA Kit (MBS453817, MyBioSource, San Diego, CA, USA), Human LIFR ELISA Kit (MBS2513036, MyBioSource, San Diego, CA, USA), or Human OSMR ELISA Kit (MBS2514091, MyBioSource, San Diego, CA, USA), respectively.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Whisker Assay, Western Blot

Journal: Nature

Article Title: IL-11 is a crucial determinant of cardiovascular fibrosis

doi: 10.1038/nature24676

Figure Lengend Snippet: a, RNA-seq of primary cardiac fibroblasts (n = 84 biologically independent samples) with or without TGFβ1 treatment (5 ng ml−1, 24 h) and Spearman’s correlation of expression changes with fibroblast activation (Supplementary Table 2). DEseq210 fold change in expression and false-discovery rate (FDR)-adjusted P values are shown. b, RNA expression in transcripts per million (TPM) of IL11RA and IL6R across 512 cell lines from the FANTOM repository12. c, Single-cell resolution of cardiac Il11 expression (more than 0 reads per cell). t-distributed stochastic neighbour embedding (tSNE) analysis28 clusters cell types of the heart. Il11 expression is highly enriched in fibroblasts. χ2 test (P = 5.7 × 10−8). d, tSNE analysis of fibroblasts alone shows highest Il-11 expression in ECM-secreting and TGFβ1-activated fibroblasts. χ2 test (P = 0.033). c, d, Cardiac cells were sequenced from n = 1 mouse, the experiment was repeated once with similar results. e, f, Representative images (chosen from 42 per condition) of cardiac fibroblasts immunostained for ACTA2, collagen I or periostin (POSTN) after a 24-h incubation without stimulus (control), TGFβ1 or IL-11 (5 ng ml−1) (e) or with TGFβ1 (5 ng ml−1) and an anti-IL-11 neutralizing antibody or an IgG control (2 μg ml−1) (f). g, Cardiac fibroblasts were seeded in collagen gel and the area of contraction determined (n = 3 biologically independent samples) after 72 h. h, Trans-well migration assay (colourimetrically quantified, n = 3 biologically independent samples, 24 h). i, Scratch assay of wound closure in a monolayer of cardiac fibroblasts (n = 5 biologically independent samples) after 24 h. g–i, Two-tailed Student’s t-test; data are mean ± s.d.; *P < 0.05; **P < 0.01.

Article Snippet: The level of IL-11, IL11RA, MMP-2, and TIMP-1 in equal volumes of cell culture medium were quantified using the following kits: Human IL-11 Quantikine ELISA kit (D1100, R&D Systems), Human IL11RA ELISA kit (LSF8919, Lifespan Biosciences), Total MMP-2 Quantikine ELISA kit (MMP200, R&D Systems), Human TIMP-1 Quantikine ELISA kit (DTM100, R&D Systems).

Techniques: RNA Sequencing Assay, Expressing, Activation Assay, RNA Expression, Incubation, Migration, Wound Healing Assay, Two Tailed Test