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. 2020 Jan 11;19:974–985. doi: 10.1016/j.omtn.2019.11.035

Figure 6.

Figure 6

SLC7A11-AS1 Prevents β-TRCP1-Mediated Ubiquitination and Degradation of NRF2

(A and B) Subcellular distribution of SLC7A11-AS1 was detected by qRT-PCR in (A) BxPC-3-Gem and (B) PANC-1 cells. U99 and MT-RNR1 were as nuclear and cytoplasmic marker, respectively (n = 3). (C) The immunofluorescence staining of SLC7A11-AS1 (red), β-TRCP1 (green), and DAPI (blue) in BxPC-3-Gem and PANC-1 cells. (D) Nuclear extracts from BxPC-3-Gem cells were incubated with biotinylated sense and antisense SLC7A11-AS1 generated in vitro, and proteins were precipitated with streptavidin beads and subjected to immunoblotting (IB) analysis with anti-TRCP1 antibody. (E) A schematic diagram of full-length SLC7A11-AS1 and its series of truncates. (F) Nuclear extracts from BxPC-3-Gem cells were incubated with biotinylated SLC7A11-AS1 truncates and antisense SLC7A11-AS1 generated in vitro, and proteins were precipitated with streptavidin beads and subjected to IB analysis with anti-TRCP1 antibody. (G) A schematic diagram of β-TRCP1 and its truncates. (H) RIP assay analysis of the interaction of β-TRCP1 and its truncates (β-TRCP1-N, β-TRCP1-N-ΔF-box, and β-TRCP1-C) with SLC7A11-AS1 in PANC-1 cells. Whole-cell expression (input) of proteins was detected by IB with indicated antibodies (left). The asterisks indicate β-TRCP1 and its truncates bands. The immunoprecipitated SLC7A11-AS1 by using anti-HA antibody was measured by qRT-PCR and represented as a fraction of input RNA (% input) prior to immunoprecipitation (right) (n = 3). (I) Effect of SLC7A11-AS1 on β-TRCP1-mediated ubiquitination of NRF2. 293T cells co-transfected with indicated plasmids were treated with MG132 (1 μM) for 12 h and subjected to immunoprecipitation (IP) with the anti-Myc antibody, followed by IB with anti-HA antibody. Whole-cell expression (input) of proteins was detected by IB with anti-FLAG or anti-GAPDH antibodies. (J) SLC7A11-AS1 prevents β-catenin proteasomal degradation. Western blot (left) analysis of nuclear β-catenin in SLC7A11-AS1-knockdown and control PANC-1 cells treated with or without MG132 (5 μM) for 24 h. Quantification of β-catenin protein (right) was normalized to the loading control (Lamin) and expressed relative to sh-Ctrl without MG132 (n = 3). **p < 0.01. ns, not significant.