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. 2019 Mar 11;47(9):4495–4508. doi: 10.1093/nar/gkz166

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — ELK-1 mono-ubiquitination requires ternary complex formation but blocks DNA binding. (A) HA.ELK-1 and mutant proteins indicated were incubated with a radio-labelled SRE probe and recombinant core^SRF. Complexes were resolved by electrophoresis and visualized by phosphor-imaging. Solid arrowheads indicate ELK-1/E74 complexes; SS indicates super-shift obtained with anti-HA antibody (lane 2). Histogram (below) shows densitometric analysis of ELK-1/DNA complexes (solid arrows) in lanes 3–9 with ELK-1 set at 100%. (B) As in (A) except that complexes were formed with a radio-labelled E74 probe; open arrowhead indicates non-specific band seen in WCE from control cells (lane 1). (C) WCEs from HEK293 cells transfected with expression vectors for His.Tyg.UBQ and HA.ELK-1 or mutants indicated were subjected to IMAC. Isolated proteins were analysed by SDS-PAGE (7.5%) and immunoblotting with an anti-HA antibody. Lower panel: WCEs analysed for ELK-1 expression. (D) ³⁵S-labelled ELK-1 was incubated alone (lane 1), with E1, E2, UBQ and UA (lanes 2–5) and with or without Nxt and an SRE oligonucleotide duplex as indicated. Reactions were separated by SDS-PAGE (7.5%) and analysed by phosphor imaging. Bracket indicates ELK-mUBQ species. Histogram (right) shows densitometry of mono-ubiquitinated species in lanes 2–5 with lane 2 set at 100%. (E) Lysate from HEK293T cells transfected with expression vectors for His.UBQ(L73P) and HA.ELK-1 was subjected to IMAC. Recombinant ELK-1, ERK2 (Supplementary Figure S5) and IMAC samples were separated by SDS-PAGE (5–20%) transferred to nitrocellulose, renatured and incubated with radio-labelled E74 probe. Lane 2′ shows longer exposure of lane 2. ELK-1 and ELK-mUBQ presence in IMAC sample was confirmed by immunoblotting with anti-HA antibody (lane 2′′).