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. 2015 Aug 20;26(12):2989–3000. doi: 10.1681/ASN.2014100978

Figure 1.

Figure 1.

Generation and characterization of CD4-Keap1-KO mice. (A) CD4-Cre mice are crossed with Keap1F/F mice to generate CD4-Keap1-KO mice. (B) Mice are genotyped to confirm the presence of the Cre and Keap1 floxed allele using CRE and floxed primers. Lanes in B represent the following: lane 1, 100-bp DNA ladder; lane 2, 324-bp internal positive control showing CD4-Cre–negative mice; lane 3, 324-bp internal positive control and 100-bp Cre showing CD4-Cre–positive mice; lane 4, 383-bp Keap1 floxed allele in Keap1F/F mice; and lane 5, 383-bp Keap1 floxed allele in CD4-Keap1-KO mice. (C) CD4-Cre–mediated deletion of exons 2 and 3 of Keap1 is further confirmed by using deletion-specific primers. Lanes in C represent the following: lane 1, 1-Kb DNA ladder; lane 2, 2954-bp WT Keap1 allele; and lane 3, 288-bp truncated Keap1 allele after deletion of exons 2 and 3. (D) Deletion of Keap1 significantly upregulates the expression of Nrf2 targets Nqo1 (P≤0.001), Ho-1 (P=0.05), and Gclc (P≤0.01) in T cells; however, there is no change in Nrf2 and Gclm mRNA levels. (E) Western blot analysis of Nrf2 and Nqo1 in nuclear and cytoplasmic fractions of T cells isolated from CD4-Keap1-KO (n=3) and Keap1F/F mice (n=3). (F) Quantification of Nrf2 and Nqo1 levels in nuclear and cytoplasmic fractions. Data represent the mean±SD. *P≤0.05; **P≤0.01; ***P≤0.001.