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. 2023 Feb 24;42(8):e112304. doi: 10.15252/embj.2022112304

Figure EV1. p53 interacts with ALDH2.

Figure EV1

  • A
    Lysates from 293T cells transfected with HA‐p53 and Flag‐ALDH2 or vector control (−) as indicated were immunoprecipitated using anti‐Flag M2 affinity gels, and bound proteins were analyzed by western blot.
  • B–D
    Lysates from HepG2 cells (B), mouse hepatocyte (C) and CCC‐HEL‐1 cells (D) were immunoprecipitated using p53 antibody, and bound proteins were analyzed by western blot.
  • E
    Lysates from 293T cells transfected with HA‐p53 and Flag‐ALDH2, or vector control (−) as indicated and treated with doxorubicin (2 ng/ml) or nutlin3α (10 μM) for 24 h were immunoprecipitated using anti‐Flag M2 affinity gels, and bound proteins were analyzed by western blot.
  • F
    Lysates from 293T cells transfected with HA‐p53 and Flag‐ALDH2, or vector control (−) as indicated and treated with or without ethanol (5 mM) for 24 h were immunoprecipitated using anti‐Flag M2 affinity gels, and bound proteins were analyzed by western blot.
  • G
    Lysates from 293T cells transfected with HA‐ALDH2 and Flag‐p53 wildtype or deletion mutants as indicated were immunoprecipitated using anti‐Flag M2 affinity gels, and bound proteins were analyzed by western blot.
  • H
    Lysates from 293T cells transfected with HA‐p53 and Flag‐ALDH2, Flag‐ALDH2‐Δ284–291, Δ312–323 were immunoprecipitated using anti‐Flag M2 affinity gels, and bound proteins were analyzed by western blot.
  • I
    Lysates from 293T cells transfected with HA‐p53 and Flag‐ALDH2, Flag‐ALDH2‐N186A, E285A, C319A were immunoprecipitated using anti‐Flag antibody, and bound proteins were analyzed by western blot.
  • J
    HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin‐resistant ALDH2‐mut312‐323 in ALDH2‐knockout cell lines. The cells were then transfected with control siRNA or p53 siRNA for 48 h and treated with ethanol (5 mM) for 0.5 h and relative levels of acetate (J, left panel) and acetyl‐CoA (J, right panel) were examined.

Data information: Data in (A–I) represent three independent experiments and (J) are from n = 3 biological independent samples. Data are the mean ± SD. P values were determined by unpaired two‐tailed Student's t tests. **P < 0.01, ***P < 0.001, n.s., not significant.

Source data are available online for this figure.