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. 2023 Jan 4;24(3):e55762. doi: 10.15252/embr.202255762

Figure 1. METTL3 protects METTL14 from degradation.

Figure 1

  • A, B
    RT‐qPCR analysis of METTL3 and METTL14 expression (A) and immunoblot analysis of METTL3 and METTL14 protein levels (B) in shRNA knockdown METTL3 cells. Data are mean ± SEM from three biological replicates and were analyzed by two‐tailed unpaired t‐test. ***P < 0.001; n.s., not significant. β‐actin was used as the loading control in the immunoblots. METTL3 or METTL14 /β‐actin densitometric ratio was recorded by ImageJ.
  • C
    Immunoblots showing total METTL14 protein levels in HA‐tagged METTL14‐overexpressing cells. The total METTL14/β‐actin densitometric ratio was recorded by ImageJ.
  • D
    Immunoblots showing the effects of cycloheximide (CHX) treatment on SK‐Cha‐1 cells. Cells with shRNA‐mediated knockdown of METTL3 (sh‐METTL3) and negative control (sh‐NC) SK‐Cha‐1 cells were exposed to 100 μg/ml CHX. The METTL14 levels dramatically decreased in sh‐METTL3 cells exposed to CHX compared with the negative control. β‐actin was used as the loading control. The METTL14/β‐actin densitometric ratio was recorded by ImageJ.
  • E
    Immunoblots showing the protein levels in the CHX assay in the METTL3 knockout cell line E14TG2a. After 4 h of exposure to CHX, METTL14 levels dramatically decreased in METTL3 knockout (METTL3‐KO) cells, whereas those in METTL3 wild‐type (METTL3‐WT) cells showed only a slight change. β‐actin was used as the loading control. The METTL14/β‐actin densitometric ratio was recorded by ImageJ.
  • F
    Immunoblots showing the protein level of METTL14 after transfection with the FLAG‐METTL3 plasmid. β‐actin was used as the loading control. The FLAG‐METTL14/β‐actin densitometric ratio was recorded by ImageJ.
  • G
    METTL14 protein levels detected under a concentration gradient of FLAG‐METTL3 plasmids. The HA‐METTL14/β‐actin densitometric ratio was recorded by ImageJ.
  • H
    Schematic diagram of METTL3 showing the different domains. MTD, methyltransferase domain.
  • I
    HA‐METTL14 Co‐IP with FLAG‐tagged METTL3 domain‐deletion mutants. β‐actin was used as the loading control.
  • J
    HA‐METTL14 protein levels detected under a concentration gradient of plasmids encoding FLAG‐tagged METTL3 domain‐deletion mutants. β‐actin was used as the loading control. The HA‐METTL14 or FLAG‐METTL3 mutants /β‐actin densitometric ratio was recorded by ImageJ.

Source data are available online for this figure.