Fig. 1

WRN depletion affects de-novo synthesis of proteins. a Pulse-labeling of shCTR and WRN-depleted HeLa cells 3 days after shRNA induction. The same number of cells were used to prepare the lysates and radiolabeled methionine incorporation (right panel) was visualized as described in the Methods section and Supplemental Information. Coomassie stained gels of total proteins (left panel) was used to confirm equal loading. Data plotted represent relative intensities ± SEM and Two-way ANOVA followed by Sidak’s multiple comparisons test from GraphPad Prism (ns, no significant difference; *** p < 0.001) of three biological replicates. b Representative Western blot showing WRN downregulation 72 h after shRNA induction. Molecular size markers (in KiloDaltons) are shown. c as in (a) using the indicated antibodies. Equal amounts of antibody used in each IP reaction was confirmed by Coomassie staining (left panel). The amount of each immunoprecipitated radiolabeled protein (red squares, right panel) in shCTR and shWRN extracts was normalized against immunoglobulin heavy chain (IgG HC) from the Coomassie-stained gel. The experiment shown is a representative of three biological replicates. d Plot of the normalized band intensities of ³⁵S generated represent relative intensities ± SEM and Two-way ANOVA followed by Sidak’s multiple comparisons test from GraphPad Prism (** p < 0.01) of three biological replicates