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The RGD-containing hexapeptide, KPRGDV, was designed based on the amino acid sequence of mouse Npnt (NCBI reference sequence NP_001025007.1).
hDPSCs were seeded into 12-well plates coated by either Npnt or the other proteins (BSA, Fn, and COL-1) at the concentration of 2 x [10.sup.4]/well and incubated at 37[degrees]C in 5% C[O.sub.2].
Confirmation of the Presence of Npnt on Polystyrene Surface.
The three matrix proteins (Npnt, Fn, and FCOL1) significantly promoted the proliferation of cells in the absence of serum at 18 h, and this effect persisted to 64 h (Figure 4).
The counting of DAPI-stained nucleus further confirmed a significant reduction of attached cell number in Npnt-RGD preincubated group, indicating adhesion of cells to the Npnt-coated substrate(s) was partly mediated by the RGD domain within Npnt (Figure 5(b)).
It is shown in Figure 6 that ITGA1 and ITGB1 were simultaneously upregulated to 1.31 [+ or -] 0.04-fold (p <0.01) and 1.87 [+ or -] 0.09-fold (p <0.01) by Npnt, respectively.
Primary antibody specific to Npnt was used to verify that the protein had undergone efficient immobilization (Figure 1).
Early observation of cell protrusions formed by actin polymerization in the FCOL1 and Npnt groups indicated potential positive effects adopted by these two proteins in directing differentiation of cells, which were echoed by the later ARS staining data.
hDPSCs had well-spread shape, with actin fibers that spanned the dimensions of cells in Npnt, Fn, and FCOL1-coated surfaces (Figure 3).
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