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. Author manuscript; available in PMC: 2012 May 13.
Published in final edited form as: J Mol Biol. 2011 Mar 21;408(4):616–627. doi: 10.1016/j.jmb.2011.03.013

Fig. 4.

Fig. 4

(a) Electrostatic surface representation of Dlar(Ig1-2). This view is related to the left view on panel c by a counterclockwise rotation of 120° along a vertical axis. Regions with negative electrostatic potential are colored red and regions with positive electrostatic potential are colored blue (scale ± 5 e/kT). (b) Electrostatic surface representation of mouse LAR(Ig1-2). Electrostatic potentials were calculated with DELPHI 46, 47. (c) Interactions between mutants of Dlar(Ig1-2) and heparin. Fragments of Dlar fused to hGH were expressed transiently in HEK293 cells. These fragments include wild-type Dlar(Ig1-2) (labeled Dlar(Ig1-2), WT), Dlar(Ig1-2) with cysteine residues at positions 52 and 219 (Dlar(Ig1-2), Cys), with asparagine residues at positions 52 and 218 to introduce N-linked carbohydrates (Dlar(Ig1-2), Asn), Dlar(Ig1) and Dlar(Ig2). The left panel shows wild-type Dlar(Ig1-2) along with its cysteine and asparagine mutants to illustrate the difference in size between the proteins upon introduction of consensus N-linked glycosylation sites in Dlar(Ig1-2). Samples were resolved by SDS-PAGE (9% gel) and fusion proteins were visualized by immunoblotting against hGH. The right panel shows the results of heparin affinity isolation assays. Conditioned media were incubated with heparin-sepharose in PBS with 1 % (v/v) Tween-20 for one hour at room temperature. Resins were washed in the same buffer and samples were resolved by SDS-PAGE (12% gel). Bound fusion proteins were visualized by immunoblotting against hGH.