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. 2021 Jan 19;11(5):1369–1385. doi: 10.1016/j.jcmgh.2021.01.007

Figure 6.

Figure 6

TRIM21 silencing protects cells from DEN-induced oxidative damage.TRIM21–/– MEFs reconstituted with vector, HA-TRIM21 WT, HA-TRIM21 LD, and HA-TRIM21 W381/383A mutant. (A) Cells were lysed in RIPA buffer with 1% SDS and subjected to WB. (B–E) Cells were treated with DEN (20 mM) for 6 hours. (B) Cells were harvested and probed for indicated proteins by WB. (C) Cells were stained with H2DCFDA and analyzed by flow cytometry. Quantification of relative H2DCFDA intensity (geometry mean of 3 repeats) is shown on the right. ∗∗∗P < .001. (D) Cells were subjected to IF with Keap1 and p62 antibodies and observed under deconvolution microscope. Cells with p62/Keap1 aggregates were counted blindly. Data shown are the averages plus SD of at least 3 countings with over 200 cells. ∗∗∗P < .001. (E) Cells were separated into Triton X-100 (1%) soluble and insoluble fractions, and 30-μg soluble proteins and corresponding volume of insoluble proteins were used for WB. The numbers below Western blot panels indicate the ratios of absolute levels of insoluble and soluble proteins, respectively.