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

. 2019 Jul 23;10:3293. doi: 10.1038/s41467-019-11149-1

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

© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 5 — VHL Phe_n+3 is associated with a more stable VHL/HIFα complex. a The structure of HIF1α-VHL complex (PDB: 1LM8) is rendered as a cartoon superimposed with a transparent surface representation. Using multiple sequence alignments for VHL and HIFα in vertebrate and invertebrate species, amino acid residues were color-coded according to conservation using the Consurf webserver. b Biotinylated HIFαOH peptides were immobilized on streptavidin- agarose beads and incubated with in vitro transcribed and translated (IVTT) HA-VHL. Streptavidin beads were pulled down (PD) and levels of HA-VHL were visualized via immunoblotting (IB). c 3xFLAG-HIFα oxygen-dependent degradation (ODD) domains were IVTT and incubated with purified HIS₆-PHD2 (181–426). Following hydroxylation (one hour), 3xFLAG-HIFα ODD domain was immobilized on protein A beads coated with anti-FLAG antibody and incubated with IVTT HA-VHL. 3xFLAG-HIFα ODD domain was immunoprecipitated (IP) and levels of HA-tagged VHL were visualized via immunoblotting (IB). Molecular weight markers (kDa) are labeled for western blots. d, e Biolayer interferometry kinetic analysis of VHL-elongin B-elongin C (VBC) complex binding to immobilized HIFα peptides. Biotinylated peptides were immobilized on streptavidin- coated biosensors and binding to VBC complex was monitored. The data was analyzed assuming a 1:1 binding model via the BLItz Pro software. d Rate plane with Isoaffinity Diagonals (RaPID) plot highlighting the kinetic parameters of VBC complex, either WT of F91Y, binding to HIFα peptides. Values represent mean of three experiments conducted with independently purified protein ± s.e.m. e The dissociation constants associated with VBC binding to HIFα peptides are shown on a linear scale. Statistical significance was assessed using a one-way ANOVA with Tukey’s post hoc test. Values represent mean of three experiments conducted with independently purified protein ± s.d. * p < 0.05, *** p < 0.005. f An idealized phylogenetic tree of metazoan evolution is depicted. Short HIF1α, HIF2α, and VHL sequences from representative species are given