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. 2018 Aug 15;38(17):e00252-18. doi: 10.1128/MCB.00252-18

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Copyright © 2018 Sekine et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 5 — OGT/HCF-1 complex is required for accumulation of NRF1 protein. (A and B) NRF1 accumulation in response to MG132 treatment in HeLa cells. HeLa cells that were transfected with control siRNA, OGT siRNA-1 (A), or OGT siRNA-2 (B) were treated with 1 μM MG132 at 72 h after transfection. Whole-cell extracts were prepared at the indicated time points after treatment with MG132. Reduction of OGT protein was verified in OGT siRNA-treated cells, and tubulin was used as a loading control. (C and D) NRF1 and NRF2 accumulation in response to a proteasome inhibitor. MDA-MB-231 cells that were transfected with control siRNA, HCF-1 siRNAs (C), or OGT siRNAs (D) were treated with 1 or 10 μM MG132 at 72 h after the transfection and cultured for another 4 h before harvest. Whole-cell extracts were prepared and subjected to immunoblot analysis with antibodies against NRF1, NRF2, HCF-1 (C), OGT (D), and tubulin. (E and F) Relative mRNA levels of NRF1 in MDA-MB-231 cells that were transfected with control siRNA, HCF-1 siRNAs (E), or OGT siRNAs (F). Values were normalized to HPRT values. Normalized values of control cells were set to 1. Averages and SD were calculated from the results of triplicate samples.