Skip to main content
. Author manuscript; available in PMC: 2015 Jan 12.
Published in final edited form as: Nat Commun. 2014 Nov 4;5:5248. doi: 10.1038/ncomms6248

Figure 6. In vitro analysis of AUF1 and HuR binding to target mRNAs and regulation of translation.

Figure 6

(a–c) Recombinant His-AUF1, MBP and MBP-HuR proteins (a) and templates for in vitro transcription of substrate RNAs, as well as transcribed RNA (b, bottom) used in biotin pull-down assays (c). Biotin pulldown was carried out using RL, RL-APP(3′UTR) or RL-TOP2A(3′UTR) (1 μg each) in the presence of 1 μg of recombinant His-AUF1 and 0, 2, 4 or 6 μg MBP-HuR before pulldown using streptavidin sepharose beads. Following SDS–PAGE, proteins were visualized by staining with Coomassie blue. (d,e) Individual and combined (ALL) recombinant proteins (d) were used in in vitro translation assays. Rabbit reticulocyte lysate reactions contained T7 RNA polymerase, 0.5 μg of the plasmids in b, expressing reporter RL, RL-APP(3′UTR) or RL-TOP2A(3′UTR) mRNAs, 0.5 μg of recombinant protein (His-AUF1 alone or in combination, MBP-HuR or MBP). Reaction components were separated with SDS–PAGE and western blot analysis was performed to detect Renilla luciferase (RL) and loading control α-Tubulin. Data are representative of three independent experiments.