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. 2011 Jun 24;6(6):e21482. doi: 10.1371/journal.pone.0021482

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Shi et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Glycosylation of endogenous ECSM2. HUVEC lysates were immunoprecipitated with anti-ECSM2 RabMAb (lanes 2 and 3) or rabbit IgG as a control (lane 1). Samples were treated with (+) or without (-) glycosidase mix, as detailed in Methods, resolved by SDS-PAGE, and immunoblotted with anti-ECSM2 RabMAb. Positions of glycosylated (mature) ECSM2, deglycosylated ECSM2, and IgG are indicated. (B) Glycosylation of ECSM2-FLAG. HEK293 cells stably expressing mouse ECSM2-FLAG were lysed and cell lysates were directly subjected to enzymatic deglycosylation reactions as described in Methods. Samples were analyzed by immunoblotting with anti-FLAG M2 mAb (lanes 1 and 2) and anti-ECSM2 RabMAb (lanes 3 and 4), respectively. Positions of glycosylated (mature) ECSM2-FLAG, and deglycosylated ECSM2-FLAG are indicated.