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. 2017 Apr 10;7(7):1890–1900. doi: 10.7150/thno.19135

Figure 6.

Figure 6

TAM activated Nrf2 through phosphorylation at ser40. A, Two or four hours after 4-OH-TAM (20 μM) incubation, nuclear and cytoplasmic distributions of Nrf2 in RL95-2 and AN3CA cells were explored by western blotting. B, Ser40 phosphorylation of Nrf2 (p-Nrf2) and total Nrf2 levels in RL95-2 and AN3CA cells after 1 hour 4-OH-TAM (20 μM) incubation were determined by western blotting. C, Nuclear and cytoplasmic distributions of p-Nrf2 in RL95-2 and AN3CA cells treated with or without 1-hour treatment of 4-OH-TAM (20 μM) were analyzed by western blotting. D, P62-pro/pGL2 or pGL2 vector were co-transfected with Nrf2 wild type (Nrf2-WT) or S40 phosphorylation deficient mutant (Nrf2-S40A) or S40 phosphorylation mimic mutant (Nrf2-S40E) or vector in AN3CA cells. Forty-eight hours later, the effects of wild type or mutated Nrf2 on SQSTM1 promoter activity were determined by luciferase activity assay. The experiments were performed in triplicate and repeated three times. Representative results were shown as Mean±SD. Asterisks indicate statistical significance (Student's T test, p<0.05). E, Forty-eight hours after Nrf2-WT or Nrf2-S40A or Nrf2-S40E or vector transfection in AN3CA cells, the expression and nuclear distribution of wild type or mutated Nrf2 and SQSTM1 protein expression were analyzed by western blotting. F, Nrf2-WT or Nrf2-S40A was transfected into AN3CA cells for 48 hours. Nuclear distributions of Nrf2-WT or Nrf2-S40A in AN3CA cells before and after 2h incubation of 4-OH-TAM (20 μM) were explored by western blotting.