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. 2016 Nov 1;25(21):1681–1690. doi: 10.1089/scd.2016.0081

FIG. 1.

FIG. 1.

Protein profile and glycan composition of cellular and exosomal Lefty in hESCs. (A) Depicts Lefty protein profile in the cytosolic extract and CM from hESCs (H7) as determined by SDS-PAGE (4%–20% acrylamide gels) and western analysis. The same approach was employed to examine Lefty's presence in the purified exosomal fractions from H7 and H9 CM and is presented in (B). For comparative purpose, the cytosolic extracts from H7 and H9 cells are included, with CD63 serving as exosomal marker. (C) The exosome-depleted CM was further tested for the presence of Lefty proteins. (D) Glycoform analysis of cellular versus exosomal Lefty in H9 hESCs. Thirty micrograms of cytosolic extracts and 5 μg of exosomal fractions were subjected to Endo-H and PNGase F treatment and analyzed by SDS-PAGE on a 10% acrylamide gel. (E) Recombinant Lefty 1 and 2 were included as control and subjected to identical digestion protocol. Cleavage products are indicated by ‘*’. CM, conditioned media; Endo-H, Endoglycosidase H; hESCs, human embryonic stem cells; PNGase F, peptide:N-glycosidase F; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.