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. Author manuscript; available in PMC: 2014 Feb 8.

Published in final edited form as: Oncogene. 2012 Sep 10;32(32):3765–3781. doi: 10.1038/onc.2012.388

Transcription factor Nrf2 contains two separate sequences in its Neh6 domain to which β-TrCP can bind.

A) COS1 cells were co-transfected with pcDNA3.1 expression plasmids encoding V5-tagged mouse Nrf2^Δ17-32 or mutants lacking SDS1, SDS2, or SDS1 and SDS2, along with pcDNA4-βTrCP1-FLAG. Empty pcDNA3.1 vector was included in the transfection mixture to normalize the amount of DNA to which cells were exposed. Following overnight transfection, the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS before whole cell lysates were prepared. An aliquot (10%) of the lysate was withdrawn as the input sample, and the remainder was used for the pull-down assay that employed an antibody against FLAG as described in Materials and Methods.

B) COS1 cells were co-transfected for 24 h with an expression vector for mouse Nrf2^Δ17-32-V5, or its mutants lacking SDSGIS³³⁸, SDSEME³⁷⁰ and DSAPGS³⁷⁸, either individually or as double deletion mutants, along with an expression plasmid for FLAG-tagged β-TrCP1. As in panel A, β-TrCP1 was pulled-down after the cells had been subjected to 16 h serum-depletion using antibodies against FLAG, and the Nrf2 mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against the V5 epitope.

C) COS1 cells were co-transfected with expression vectors for a YFP-Neh6 fusion protein, or YFP-Neh6 protein lacking SDSGIS³³⁸, SDSEME³⁷⁰ or DSAPGS³⁷⁸, or combinations thereof, along with an expression plasmid for FLAG-tagged β-TrCP1. The Neh6 domain mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against GFP.

Transcription factor Nrf2 contains two separate sequences in its Neh6 domain to which β-TrCP can bind.

A) COS1 cells were co-transfected with pcDNA3.1 expression plasmids encoding V5-tagged mouse Nrf2^Δ17-32 or mutants lacking SDS1, SDS2, or SDS1 and SDS2, along with pcDNA4-βTrCP1-FLAG. Empty pcDNA3.1 vector was included in the transfection mixture to normalize the amount of DNA to which cells were exposed. Following overnight transfection, the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS before whole cell lysates were prepared. An aliquot (10%) of the lysate was withdrawn as the input sample, and the remainder was used for the pull-down assay that employed an antibody against FLAG as described in Materials and Methods.

B) COS1 cells were co-transfected for 24 h with an expression vector for mouse Nrf2^Δ17-32-V5, or its mutants lacking SDSGIS³³⁸, SDSEME³⁷⁰ and DSAPGS³⁷⁸, either individually or as double deletion mutants, along with an expression plasmid for FLAG-tagged β-TrCP1. As in panel A, β-TrCP1 was pulled-down after the cells had been subjected to 16 h serum-depletion using antibodies against FLAG, and the Nrf2 mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against the V5 epitope.

C) COS1 cells were co-transfected with expression vectors for a YFP-Neh6 fusion protein, or YFP-Neh6 protein lacking SDSGIS³³⁸, SDSEME³⁷⁰ or DSAPGS³⁷⁸, or combinations thereof, along with an expression plasmid for FLAG-tagged β-TrCP1. The Neh6 domain mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against GFP.