Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke–induced emphysema in mice
J. Clin. Invest. Tirumalai Rangasamy, et al. 114:1248 doi:10.1172/JCI21146 [
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Figure 6Activation of Nrf2 in CS-exposed
Nrf2+/+ lungs. (
A) EMSA to determine the DNA binding activity of Nrf2. For gel shift analysis, 10 μg of nuclear protein from the lungs of air-and CS-exposed mice was incubated with the labeled human NQO1 ARE sequence and analyzed on a 5% non-denaturing polyacrylamide gel. For supershift assays, the labeled NQO1 ARE was first incubated with 10 μg of nuclear extract and then with 4 μg of anti-Nrf2 antibody for 2 hours. Nuclear protein of
Nrf2+/+ lungs showed increased binding to the ARE-containing sequence (lower arrow) after CS exposure, with a supershifted band caused by preincubation with anti-Nrf2 antibody, thus confirming the binding of Nrf2 to the ARE sequence (upper arrow). Ra–IgG1, rabbit IgG1. (
B) Nuclear accumulation of Nrf2. Western blot analysis with anti-Nrf2 antibody showed the nuclear accumulation of the transcription factor Nrf2 in the lungs of
Nrf2+/+ mice in response to CS exposure (lanes 1 and 3: air-exposed
Nrf2–/– and
Nrf2+/+ mice, respectively; lanes 2 and 4: CS-exposed
Nrf2–/– and
Nrf2+/+ mice, respectively; lamin B1 was used as the loading control). Western blot analysis was carried out 3 times with the nuclear proteins isolated from the lungs of 3 different air- or CS-exposed
Nrf2+/+ and
Nrf2–/– mice.
