Molecules and Cells

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Fig. 1.

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Fig. 1. (A) Scheme of development of novel TNFR2 synthetic peptides. S, signal sequence; PLBS, polypeptide ligand binding site; HI, homodimer interface. (B and C) U937 cells were incubated for 30 min with various TNFR2-peptides (TNFR2-SKEE and TNFR2-SKE) and variant (TNFR2var1-IKE) or various concentrations of TNFR2-SKE, followed by treatment with 50 ng/ml human TNF-α for 10 min in the presence or absence of each peptide. The interaction between TNFR2 and TRAF2 was analyzed using in situ PLA. The graph indicated the intensity of interaction between molecules mentioned above. PLA signals in the cell population (n = 5) were quantified using the NIS Elements analysis. The average number of rolling-circle products (RCPs) per cell ± SD is shown. ***p < 0.001. Nuclei were stained with DAPI (blue fluorescence). Magnification, ×600; scale bars = 10 μm. (D) Three different cells (NIH3T3 mouse fibroblasts, HaCaT human keratinocytes, and human dermal fibroblasts) were seeded in 35-mm dishes and two different monocytic cells (U937 and THP-1) were maintained in culture dishes. After serum starvation for 6 h, the cells were treated with TNFR2-SKE (100 μM), human TNF-α (25 or 50 ng/ml), or mouse TNF-α (25 ng/ml) for 15 min as described in Materials and Methods. Whole cell lysates were subjected to SDS-PAGE and analyzed using western blotting with the indicated antibodies. Band intensity was quantified using ImageJ software.
Mol. Cells 2019;42:151~160
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