Fig. 5: GR80 interacts with RQC factors and induces ribosomal stalling. | Nature Communications

Fig. 5: GR80 interacts with RQC factors and induces ribosomal stalling.

From: Stalled translation by mitochondrial stress upregulates a CNOT4-ZNF598 ribosomal quality control pathway important for tissue homeostasis

Fig. 5

a Western blot analysis of indicated proteins in HEK293T cells overexpressing FLAG-GR80. b Immunoprecipitation analysis testing potential interaction between FLAG-GR80 and RQC factors (ASCC3, ZNF598, RPS10, EDF1, 4EHP, GIGYF2, CNOT4). Calmodulin serves as a negative control. c Immunoblots showing effect of GR80 on translational readthrough of the GFP-P2A-FLAG-K20-P2A-mCherrry stall reporter in normal and ZNF598 KO HEK293T cells. d Cell extracts from control (left panels) or FLAG-GR80-overexpressing HEK293T cells (right panels) were fractionated on 10-50% sucrose gradients. A total of 11 fractions were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. e Cell extracts from control (left panels) or FLAG-GR80-overexpressing HEK293T cells (right panels) were incubated with RNase A (10 μg/ml) for 30 min and fractionated on 10-50% sucrose gradients. Polysome profiles were derived from RNase-digested lysates of HEK293T cells and GR80 overexpressing HEK293T cells. 11 fractions were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. Representative blot is from at least three independent experiments with similar results. Source data are provided as a Source Data file.

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