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. 2018 Aug 24;37(18):e98358. doi: 10.15252/embj.201798358

Figure 2. TRIM16 regulates the p62‐KEAP1‐NRF2 system.

Figure 2

  • A
    Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies.
  • B
    Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies.
  • C
    Left panel, Western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies. Right panel, densitometric analysis (mean ± SD) of protein band intensity relative to actin, n = 3, ***P < 0.0005 (Student's unpaired t‐test).
  • D–G
    (D, F) HEK293T cells were either transiently transfected with an empty vector or a Flag‐tagged‐TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies. L.E, low exposure; H.E, high exposure. (E, G) Densitometric analysis (mean ± SD) of protein band intensity relative to actin, n = 3, *P < 0.05 (Student's unpaired t‐test).
  • H
    WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 μg/ml) for the indicated period and probed with different antibodies as indicated. The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X‐ray film.
  • I
    Quantification of NRF2, p62, and KEAP1 band intensities relative to actin. n = 3, **P < 0.005, *P < 0.05 (Student's unpaired t‐test).
  • J
    Immunoprecipitation (IP) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 (10 μM, 4 h). *The panel (J) here, Fig EV2H, and Appendix Fig S3H are part of same blots; hence, the input for TRIM16 is same.
  • K
    The domain organization map of TRIM16 and deletion constructs cloned as Myc‐tagged proteins.
  • L, M
    IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (L) absence and (M) presence of MG132 (10 μM, 4 h).
  • N
    IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates. Mr, molecular weight marker.
  • O
    Co‐IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates.
  • P
    IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates.

Source data are available online for this figure.