Journal: Molecular Vision

Article Title: Analysis of interleukin-20 receptor complexes in trabecular meshwork cells and effects of cytokine signaling in anterior segment perfusion culture

doi:

Figure Lengend Snippet: Schematic of interleukin-20 receptor complex before and after activation by IL-20, -19, or -24. The type I receptor is composed of the interleukin-20RA (IL-20RA) and IL-20RB proteins while the type II receptor contains the IL-22RA1 and IL-20RB proteins [ , , ]. Upon cytokine binding, Jak1 binds near the transmembrane region of IL-20RA and IL-22RA1. Tyk2 is proposed to bind to IL-20RB based on sequence homology between IL-20RB and interferon-γ receptor 1 (IFNγR1) . Binding of the cytokine results in closer juxtaposition of Jak1 and Tyk2, leading to autophosphorylation of the tyrosine kinases, phosphorylation of IL-20RA, and recruitment and phosphorylation of signal transducer and activator of transcription (STAT)-3, STAT-1, and possibly, STAT-5 [ , ]. The STATs can then translocate to the nucleus to activate transcription.

Article Snippet: The membranes were then probed with the following signal transducer and activator of transcription (STAT) antibodies (all antibodies from Cell Signaling Technology Inc., Danvers, MA, unless stated otherwise): phospho-tyrosine701 STAT-1 (mouse monoclonal; ThermoFisher, 33–3400), total STAT-3 (mouse monoclonal; 9139), phospho-serine727 STAT-3 (rabbit polyclonal; 9134), phospho-serine705 STAT-3 (rabbit polyclonal; 9131), total STAT-5 (rabbit polyclonal; 25,656), and phospho-TyrosineY694 (mouse monoclonal; 9356).

Techniques: Activation Assay, Binding Assay, Sequencing

Journal: Molecular Vision

Article Title: Analysis of interleukin-20 receptor complexes in trabecular meshwork cells and effects of cytokine signaling in anterior segment perfusion culture

doi:

Figure Lengend Snippet: Western immunoblot analysis of STAT phosphorylation. Western immunoblot analysis of signal transducer and activator of transcription (STAT)-1 Tyr701, STAT-3 Ser727, STAT-3 Tyr705, and STAT-5 Tyr694 phosphorylation in ( A ) normal human trabecular meshwork cells or ( B ) normal human dermal fibroblasts treated with the indicated interleukins for 15 min. Total STATs were used as loading controls. The arrow points to the upper phosphorylated band. C: Densitometry of the phosphorylated Ser727 STAT-3 band. Trabecular meshwork, n=6 technical replicates using four independent cell strains; dermal fibroblasts, n=3 technical replicates from three cell strains. * p<0.05; ** p<0.005 with one-way ANOVA.

Article Snippet: The membranes were then probed with the following signal transducer and activator of transcription (STAT) antibodies (all antibodies from Cell Signaling Technology Inc., Danvers, MA, unless stated otherwise): phospho-tyrosine701 STAT-1 (mouse monoclonal; ThermoFisher, 33–3400), total STAT-3 (mouse monoclonal; 9139), phospho-serine727 STAT-3 (rabbit polyclonal; 9134), phospho-serine705 STAT-3 (rabbit polyclonal; 9131), total STAT-5 (rabbit polyclonal; 25,656), and phospho-TyrosineY694 (mouse monoclonal; 9356).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Cellular interplay via cytokine hierarchy causes pathological cardiac hypertrophy in RAF1 -mutant Noonan syndrome

doi: 10.1038/ncomms15518

Figure Lengend Snippet: ( a ) Schematic of direct and transwell co-culture experiments. ( b ) Neonatal WT CMs were co-cultured for 3 days with CD31 + cells from postnatal day 4–7 Tie2-L613V or control hearts. Left: Representative immunofluorescence staining for CD31 (magenta), cardiac troponin T (green) and DAPI (blue) in co-cultures (original magnification, × 200, scale bar, 50 μm). Arrows indicate individual CMs that were among those assessed by ImageJ. Right: Quantification of one of the three independent experiments with similar results. ( c ) Left: Transwell assays. CD31 + cells were seeded in the upper chamber and sizes of neonatal WT CMs (green) in the bottom chamber were assessed using ImageJ (original magnification, × 400, scale bars, 50 μm). Right panel: Quantification of one of three independent experiments with similar results (mean±s.e.m.; 300 CMs counted per group; ** P <0.005, *** P <0.001, two-tailed Mann–Whitney test). ( d ) Conditioned media were collected from CD31 + cultures 2 days after a media change, and TNF and IL6 levels were measured by Luminex assay (mean±s.e.m.; n =4 each group; ** P <0.005, * P <0.05, two-tailed Student's t -test). ( e ) Tnf mRNA levels in cultured ECs, assessed by qRT-PCR (mean±s.e.m.; n =4 each group; ** P <0.005, two-tailed Student's t -test). ( f , g ) CMs and CD31 + cells were co-cultured overnight, and then subjected to either ( f ) anti-TNF antibody (MP6-XT22, 1 ng ml −1 ), or ( g ) anti-IL6 neutralizing antibody (MP5-20F3, 10 ng ml −1 ) or isotype control (IgG1) in the presence or absence of excess recombinant TNF (1 ng ml −1 ) or IL6 (25 ng ml −1 ), as indicated. Quantification is shown for one of three independent experiments with similar results (mean±s.e.m.; 300 CMs counted per group; ** P <0.005, *** P <0.0001, Dunn's post hoc test when ANOVA (Kruskal–Wallis test) was significant; ## P <0.005, two-tailed Mann–Whitney test). Note that either exogenous TNF or IL6 reverses the inhibitory effect of anti-TNF antibody. ( h ) Heart lysates from Tie2-L613V or control mice were analysed by immunoblotting with the indicated antibodies. STAT3 and STAT5 levels serve as loading controls.

Article Snippet: Antibodies used for immunoblots included: anti-ERK2 (7 ng ml –1 , Clone D-2; Santa Cruz Biotechnology Inc.), and anti-SERCA2 (Clone D51B11; #9580), anti-phospho- p44/42 MAPK (#9101), -phospho-MEK1/2 (#9121), -phospho-AKT (S473, #9271), -phospho-AKT (T308, Clone 244F9, #4056), -AKT1 (Clone C73H10, #2938), -STAT3 (#9132), -phospho- STAT3 (Y705, #9131), -STAT5 (#9163), -phospho-STAT5 (Y694, #9351; all at 1:1,000 dilutions from Cell Signalling Technology).

Techniques: Co-Culture Assay, Cell Culture, Control, Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY, Luminex, Quantitative RT-PCR, Recombinant, Western Blot