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. 2013 Nov 7;8(11):e80387. doi: 10.1371/journal.pone.0080387

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© 2013 Bergiers 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) Co-precipitation assays involving mutant forms of Hoxa2. HEK293T cells were cotransfected with expression vectors for FLAG-tagged Hoxa2 (FLAG-Hoxa2wt), FLAG-tagged Hoxa2^KQN-RAA (FLAG-Hoxa2KQN-RAA), FLAG-tagged Hoxa2^WM-AA (FLAG-Hoxa2WM-AA), GST-tagged RCHY1 and GST proteins, and treated with the proteasome inhibitor MG132. Forty-eight hours after transfection, cell lysates were subjected to western blotting (input) and protein interactions were verified by co-precipitation on glutathione beads directed toward the GST tag. Eluted proteins were analysed by western blotting using the M2 anti-FLAG antibody (CoP). (B) Amino acid substitutions in the Hoxa2 homeodomain abolish the Hoxa2-mediated degradation of RCHY1. HEK293T cells were transfected with expression vectors for FLAG-tagged RCHY1 (FLAG-RCHY1) and GST-tagged Hoxa2 (GST-Hoxa2wt, GST-Hoxa2KQN-RAA, GST-Hoxa2WM-AA) proteins. Cells were then treated for proteasome inhibition (MG132) and compared to untreated controls (DMSO) for the decay of FLAG-RCHY1 revealed by western blot detection. Detection of beta-actin was used as a protein load control.