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. 2008 Apr 21;181(2):309–320. doi: 10.1083/jcb.200712068

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Copyright © 2008, The Rockefeller University Press

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Figure 6. — Time course for GT1 reglucosylation of HA. (A) HA was transiently overexpressed in wild-type or MI8-5 cells. Transfected cells were pulse labeled in the presence of 4 mM DTT for 5 min and chased under nonreducing conditions for the indicated times. 0.5 mM DNJ was present throughout the experiment for lanes 6 and 12. Radiolabeled HA was isolated by either immunoprecipitation with HA antisera or GST-calreticulin (GST-CRT) pulldown and resolved via 7.5% SDS-PAGE. (B) Quantifications of A. Fraction bound by GST-calreticulin pulldown versus total immunoprecipitated HA, normalized to maximum reglucosylation observed in the presence of DNJ. (C) HA was transiently overexpressed as in A. Transfected cells were pulse labeled in the presence of 4 mM DTT for 30 min, washed extensively to remove the reductant, and chased under nonreducing conditions for 30 min before alkylation and immunoprecipitation. 0.5 mM DNJ was present throughout the starvation, pulse, and chase period as indicated. Radiolabeled HA was immunoprecipitated with polyclonal HA, calnexin (αCNX), or calreticulin (αCRT) antisera. Radiolabeled calnexin (closed triangle) and calreticulin (open triangle) are indicated. Samples were resolved via 7.5% nonreducing (top) and reducing (bottom) SDS-PAGE.