Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2010 May 12;5(5):e10600. doi: 10.1371/journal.pone.0010600

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Coulombe 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.

PMC Copyright notice

(A) Expression vectors carrying cDNAs corresponding to the 1.8 kb (with and without a MYC-tag), the extended 1.8 kb (MYC-tagged and non-tagged), the 5.0 kb-Hoxa6-FLAG/a5-MYC, the 5.0 kb-Hoxa5-MYC and the 9.5 kb Hoxa6-FLAG/a5-MYC transcripts were tested in vitro using a coupled transcription/translation system. A [S³⁵]-radiolabeled protein corresponding to a ∼38 kD genuine HOXA5 protein is translated from all vectors. The larger HOXA5 isoform of ∼50 kD is only translated from the extended 1.8 kb cDNA version. The 5.0 kb-Hoxa6-FLAG/a5-MYC and the 9.5 kb Hoxa6-FLAG/a5-MYC vectors also produce a ∼36 kD band likely corresponding to the putative HOXA6 protein. Translation of the HOXA5 and HOXA6 proteins from the 9.5 kb Hoxa6-FLAG/a5-MYC vector is weakly detected and a longer exposure is shown with arrows to indicate the position of both proteins. (B) The HEK293 cells were transfected with the MYC-tagged version of the 1.8 kb and extended 1.8 kb expression vectors and with the 5.0 kb-Hoxa6-FLAG/a5-MYC, the 5.0 kb-Hoxa5-MYC and the 9.5 kb Hoxa6-FLAG/a5-MYC vectors. In parallel, control plasmids expressing either the green fluorescent protein (GFP ctl), the 1.8 kb Hoxa5-MYC in the antisense orientation or the pMEK1-MYC-FLAG plasmid (MYC/FLAG ctl) were transfected. Protein lysates were western-blotted with anti-MYC or anti-FLAG antibodies. Solely the 1.8 kb-MYC vector produces a genuine HOXA5 protein whereas the HOXA6 protein is only detected with the 5.0 kb-Hoxa6-FLAG/a5-MYC plasmid. GAPDH was used as a loading control. (C) RNA expression of each expression vector transfected in HEK293 cells was tested by RT-PCR. A 305 bp fragment is observed in RNA samples from HEK293 cells transfected with the Hoxa5 cDNA vectors. No expression is detected in the GFP ctl specimen.