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FIG. 3. xARH interacts with the LDL and VTG receptors. A, total RNA was isolated from embryos at different stages and xARH expression analyzed by reverse transcriptase-PCR. Ornithine decarboxylase (ODC) served as the loading control. Reaction without reverse transcriptase added (–RT) was used as the negative control. B, GST pull-down assays showing interaction between xARH and the LDL and VTG receptors. In vitro translated and 35S-Met labeled xARH-N terminus (residues 1–212) or VTGR was mixed with purified GST-LDLR or GST-xARH. The GST fusion proteins and bound proteins were pulled down with glutathione-Sepharose beads and analyzed by SDS-PAGE. The GST fusion proteins were visualized by Coomassie Blue staining. The in vitro translated proteins were detected by exposure to a PhosphorImager screen. C, the cytoplasmic tails of the LDLR and VTGR were translated in vitro and labeled by 35S-Met incorporation. In mutants (mt), the NPVY motifs were changed to NPVA. Purified GST-xARH fusion protein from E. coli was mixed with the cytoplasmic tails and glutathione-Sepharose beads. The beads were spun down and boiled in SDS loading buffer (pellet). Both the supernatant (S) and pellet (P) fractions were resolved by SDS-PAGE. The cytoplasmic tails were visualized by exposure to a PhosphorImager screen. The GST-xARH protein was detected by Coomassie Blue staining. Image published in: Zhou Y et al. (2003) Copyright © 2003. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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