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. 2016 May 2;213(5):859–875. doi: 10.1084/jem.20151208

Figure 3.

Figure 3.

Gankyrin increases the expression of antioxidative enzymes. (A and B) qRT-PCR analysis was performed for SOD1, SOD2, ANT, CAT, Gpx, GCLC, GCLM, HO-1, NQO1, and AKR family members and gankyrin in MHCCLM3 and SMMC7721 control and gankyrin knockdown cells. The data are the mean ± the SEM of three independent experiments. (C) qRT-PCR analysis was performed to detect SOD1, SOD2, ANT, and Gpx expression in SMMC7721 cells that were transiently transfected with gankyrin-pcDNA3.1A vectors with or without H2O2 stimulation. The data are expressed as the mean ± SEM of three independent experiments. (D) Western blot analysis showed the levels of NQO1, HO-1, and GCLM protein in SMMC7721-con, SMMC7721-siGank, MHCCLM3-con, and MHCCLM3-siGank cells. The data shown are representative of three independent experiments. (E) Western blotting analysis showed the levels of AKR1B10 and AKR1C3 protein in gankyrin-knockdown or overexpressing SMMC7721 cells. The data shown are representative of three independent experiments. (F) Gankyrin influenced ARE luciferase reporter activity in HCC cells. MHCCLM3 and SMMC7721 cells with different gankyrin levels were transiently transfected with an ARE luciferase reporter vector or the control plasmid pRL-TK for 48 h. The cells were harvested and gankyrin reporter activities were detected. The results are means ± SEM. n = 3. *, P < 0.05; **, P < 0.01. Representative results from three experiments are shown. (G) Nrf2 protein levels were detected in three HCC cells by Western blot analysis. The data shown are representative of three independent experiments. (H) Nrf2, Keap1, and Cullin3 protein levels in SMMC7721-con, SMMC7721-ovGank cells, MHCCLM3-con, and MHCCLM3-siGank cells were detected by Western blot. The data shown are representative of three independent experiments. (I) The ubiquitination status of Nrf2 in gankyrin-knockdown and control cells. Whole-cell lysates were immunoprecipitated with an anti-Nrf2 antibody or control immunoglobulin G and analyzed by Western blot with antiubiquitin antibody. The data shown are representative of three independent experiments.