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. 2019 Dec 12;21(2):e48597. doi: 10.15252/embr.201948597

Figure 2. LSD1 R838 methylation promotes the stabilization of LSD1.

Figure 2

  • A
    Methylation of LSD1 was assessed in MCF10A, MCF7, and MDA‐MB‐231 cells by immunoblotting using anti‐LSD1 R838me2a.
  • B, C
    Protein and relative mRNA expression levels of LSD1 in MDA‐MB‐231 cells transfected with CARM1 shRNAs or control vector were assessed by Western blotting (B) and qPCR analysis (C) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t‐test).
  • D
    MDA‐MB‐231 cells were treated with the indicated amounts of MS049 for 48 h, LSD1 R838me2a and LSD1 levels were assessed by Western blotting using indicated antibodies.
  • E
    MDA‐MB‐231 cells were treated with increasing amounts of MS049 for 48 h in the presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting.
  • F
    MDA‐MB‐231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h, and the cell lysates were analyzed by Western blotting.
  • G
    MDA‐MB‐231 cells were transfected with CARM1 shRNA#2 or control vector, and then treated with CHX (100 μg/ml) for the indicated time; LSD1 protein level was examined by Western blotting.
  • H
    MDA‐MB‐231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti‐LSD1 antibody and then analyzed by immunoblotting.
  • I
    MDA‐MB‐231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h. The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti‐LSD1 antibody.
  • J
    HEK‐293T cells that transfected with LSD1 WT or LSD1 R838A was treated with CHX for the indicated time. LSD1 was examined by Western blotting analysis.
  • K
    HEK‐293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti‐Flag antibody and then subjected to immunoblotting.
  • L
    HEK‐293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h. Co‐IP assay was performed using anti‐Flag and then subjected to Western blotting.

Source data are available online for this figure.