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

Figure 2. The ubiquitin–proteasome system regulates METTL14 protein stability.

Figure 2

  1. METTL14‐GFP Co‐IP assay in HEK293T cells. Silver staining identified the specific bands (red arrow) for METTL14‐IP (black arrow).
  2. GO analysis of the METTL14 interactome.
  3. Co‐IP of HA‐METTL14 with the proteasome subunit PSMD3‐myc in HEK293T cells.
  4. METTL14 protein levels increase significantly with increasing MG132 concentration. The FLAG‐METTL14/β‐actin densitometric ratio was recorded by ImageJ.
  5. METTL14 ubiquitination, as detected by IP of METTL14 with anti‐FLAG antibody and immunoblotting with anti‐Ub antibody. The accumulation of Ub and METTL14 was confirmed in whole‐cell lysates. β‐actin was used as the loading control.
  6. Immunoblot analysis of FLAG‐METTL14, LC3‐II, and β‐actin in HEK293T cells exposed to the autophagy inhibitor Baf‐A1 and activator Torin 1, respectively. The FLAG‐METTL14 or LC3‐II /β‐actin densitometric ratio was recorded by ImageJ.
  7. Dot blots of m6A levels in the presence of MG132 in 293 T, RBE, SK‐Cha‐1, and SK‐N‐SH cells.

Source data are available online for this figure.