ARPE-19 or human Muller cell line (MIO-M1 were starved for 2 hours and cultured in DMEM supplemented with IFNγ for 15, 30, 60, 90 minutes in the presence or absence of AG-490 or the protein synthesis inhibitor, cycloheximide. Blots were probed with rabbit polyclonal IL-27 antibody or pSTAT1, IRF-1, IRF-8, β-Actin specific antibodies. Pre-immune serum was used in parallel as controls and signals ...

Authors: Charles Egwaugu et al
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This protocol is for histology and immunohistochemistry.

Authors: Charles Egwuagu et al
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This protocol describes the isolation of naive human T cells and culture conditions that result in the development of IL-17 producing helper Th17 cells. The addition of IL-23 or IL-1 is essential for this process as is T cell receptor activation.

Authors: Rene de Waal Malefyt and Katia Boniface
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Border cell migration in the Drosophila egg chamber is a powerful, genetically tractable model to study cell invasion[1]. Studying border cell migration in Drosophila has allowed identification and characterization of several genes needed for cell migration and their guidance. The big drawback in studying border cell migration has been lack of success culturing stage 9 egg chambers and so border c...

Authors: Adam Cliffe, Minna Poukkula and Pernille Rørth
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Selectin-IgM chimeric proteins are commonly used to detect selectin ligands on tissue sections or cultured cells. There is no report to detect the ligands on membrane blot. Here we describe about ligand blotting using mouse E-selectin-IgM and L-selectin-IgM. E-selectin-IgM blotting is easier than L-selectin IgM blotting probably because of the higher affinity of E-selectin and its ligand.

Authors: Junya Mitoma and Xingfeng Bao
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To analyze glycan function, enzyme digestion is one of the easiest method. We successfully demonstrated the elimination of N-glycans, O-glycans and heparan sulfate on lymph node frozen section by N-glycanase (N-glycosidase F), O-sialoglycopeptidase, and heparitinases, respectively. N-glycanase treatment usually needs denaturing condition such as SDS treatment, but this can be substituted with ace...

Authors: Junya Mitoma, Xingfeng Bao and Minoru Fukuda
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Methods to investigate protein-protein interactions in mammalian cells are limited by the use of single reporter functions [1-5]. Such assays are susceptible to numerous intrinsic variables in the level of transfection efficiency, transcription and translation. We have developed assay systems based on enzymatic and fluorescence activities that determine the efficiency of protein-protein interactio...

Authors: Talat Nasim and Richard Trembath
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Cell renewal is essential to maintain tissue homeostasis and repair throughout the life of multicellular organisms. These functions rely on the presence of stem cells that have the potential to differentiate into specialized cell types as well as the capacity for long-term self-renewal. Adult organs therefore need long-life reservoirs of somatic stem cells to ensure the turnover of the tissue. The...

Authors: Juan-Jose Ventura
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It has been shown that signaling via phosphorylation happens within seconds after growth factor stimulation in eukaryotic cells. However, these very early signaling events have so far not been analyzed by mass spectrometry (MS)-based proteomics. Here we report the development of an automated system which allows quantitative proteomic assessment of very early cellular signaling events (qPACE). Our ...

Authors: Joern Dengjel et al
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