Fig. 5. PHD1 interacts with LRS and controls LRS stability.

a HEK293T cells were transfected with myc-LRS, myc-LRS and flag-PHD1^WT or myc-LRS and flag-PHD1^MUT. Cell lysates were immunoprecipitated with anti-flag antibody. Co-precipitation of myc-LRS was determined by western blot analysis using an anti-myc antibody. The figure shows a representative experiment. b In vitro hydroxylation assay using ³H-labeled in vitro translated LRS (gray bar) or HIF1α (white bar; positive control) in the presence of affinity purified PHD1. c HEK293T cells were transfected with myc-LRS or myc-LRS and flag-PHD1^WT and treated with DMOG or amino acid starved for 4 h. Cell lysates were immunoprecipitated with anti-flag antibody. Co-precipitation of myc-LRS was determined by western blot analysis using an anti-myc antibody. The figure shows a representative experiment. d Representative picture (left panel) and quantification (right panel) of western blot analysis of LRS protein levels in WT (black line) and PHD1^KO (red line) myotubes exposed to cycloheximide (Chx) for different amounts of time. Dots represent the mean from 2 independent experiments. e Time-course analysis of LRS protein levels in WT (black line) and PHD1^KO (red line) myotubes exposed to amino acid starvation. Dots represent the mean from three independent experiments. f Time-course analysis of LRS protein levels in WT (black line) and PHD1^KO (red line) myotubes exposed to hypoxia (1% oxygen). Dots represent the mean from 3 independent experiments. Statistics: two-way ANOVA (in e and f, interaction effects are indicated) with a a t test (d) (*p < 0.05; **p < 0.01; ***p < 0.001; ns not significant). Bar graphs and line graphs represent mean ± SEM (error bars). Data are presented as fold change to WT 0 h (d, e, f). See also Supplementary Fig. 5. Source data are provided as a Source Data file.