FIG. 2.

p62 upregulated the Nrf2 signaling pathway. (A) p62 regulates the transcriptional activity of Nrf2. Different amounts of the expression plasmids of p62-WT or p62-M were transfected into HEK293 cells along with the NQO1-ARE promoter firefly luciferase and Renilla luciferase as an internal control. As a positive control, cells were treated with known inducers of Nrf2, tBHQ, or SF overnight. Thirty-six hours posttransfection, both firefly and Renilla luciferase activities were measured. (B) Upregulation of Nrf2 downstream genes by p62. HEK293 cells were transfected with either the expression plasmid for vector, p62-WT, or p62-M. Forty-eight hours posttransfection, total mRNA was extracted using TRIzol, and qRT-PCR was performed to measure the mRNA levels of Nrf2, Keap1, HO-1, NQO1, GLCM, and MRP2. Values were normalized to GAPDH, and samples were done in triplicates. Data represented are the mean ± standard deviation (SD). (C) An increase in p62 caused an increase in Nrf2 protein levels. HEK293 cells were transfected with an expression vector for vector, p62-WT, or p62-M. Cell lysates were collected and subjected to immunoblot analysis using the following antibodies: anti-Nrf2, anti-myc (p62), anti-NQO1, and anti-β-actin.