Figure 4 | Scientific Reports

Figure 4

From: A Designed Angiopoietin-1 Variant, Dimeric CMP-Ang1 Activates Tie2 and Stimulates Angiogenesis and Vascular Stabilization in N-glycan Dependent Manner

Figure 4

Dimeric CMP-Ang1 has different glycan pattern dependent on cell line.

(A) Serum-starved HUVECs were treated with 500 ng/ml of CA1-3 expressed by either HEK293 or CHO cells for 15 min and then the phosphorylation of Tie2 was detected by immunoprecipitation and immunoblotting. The relative ratio of phospho-Tie2 (pTie2) to Tie2 in CB-treated cells is arbitrarily given as 1. Each graph represents the mean ± SD of three experiments. *p < 0.05 versus CB. (B) Analysis of CA1-3 expressed by either HEK293 or CHO cells after treatment of PNGase F to remove N-glycosylation. Reduced protein samples were separated by SDS-PAGE and were stained with Coomassie blue. (C) Crystal structure of FLD in Ang1. The N-glycosylation site, asparagine at 106 residue, is colored in red and the Tie2 binding site is colored in cyan. (D) Analysis of N-glycan of CA1-3 produced from HEK293 and CHO cells. Five glycans with the highest NAPI (normalized absolute peak intensity) values for both CHO and HEK293 cells are shown. Composition represents Hex_HexNAc_Fuc_NeuAc. (E) Correlation of glycans in CA1-3 between HEK293 and CHO cells. R2 value is indicated.

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