Fig. 4.

NRF1 Lys230 is responsible for SIAH2-mediated NRF1 ubiquitination and degradation under hypoxia. a Determination of the Lysine 230 of NRF1 is responsible for SIAH2-induced degradation. Quantification of NRF1 protein levels is shown below. b SIAH2 induces degradation of wild-type NRF1 in a dosage-dependent manner but has no effect on NRF1-K230R. c SIAH2 induces ubiquitination of NRF1 but not NRF1-K230R. d Purified SIAH2 promotes ubiquitination of bacterially expressed wild-type NRF1 but not NRF1-K230R in vitro. e Co-immunoprecipitation of exogenously expressed Myc-NRF1 or Myc-NRF1-K230R with Flag-SIAH2^RM. f Hypoxia-induced ubiquitination of NRF1 is abolished by expression of the NRF1-K230R mutant in vivo. g HeLa cells were transiently transfected with wild-type NRF1 or NRF1-K230R for 12 h, and then incubated under normoxia or hypoxia for an additional 36 h. Cells were harvested and analyzed by immunoblotting with the indicated antibodies. Quantification of NRF1 protein levels is shown below. For all panels, error bars indicate s.d., n = 3 biological replicates. Data were compared with two-tailed paired ratio t-tests