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. 2023 Jan 4;42(4):e111549. doi: 10.15252/embj.2022111549

Figure EV2. HERC3 blocks β‐TrCP‐mediated ubiquitination and proteasomal degradation of YAP/TAZ.

Figure EV2

  1. Western blots showing relevant protein levels in Fig 2B.
  2. HERC3 does not affect the protein levels of β‐TrCP1 and β‐TrCP2. HEK293T cells were transfected with expression plasmids for Myc‐HERC3, Myc‐HERC3‐CA, FLAG‐MM1, FLAG‐β‐TrCP1, and FLAG‐β‐TrCP2 as indicated. Cells were harvested 24 h post‐transfection and subjected to Western blotting analysis with FLAG, Myc, and β‐actin antibodies.
  3. HERC3 and HERC3‐CA attenuate β‐TrCP‐mediated ubiquitination of YAP. HEK293T cells were transfected with expression plasmids for His‐Myc‐ubiquitin and FLAG‐YAP, HA‐β‐TrCP, HA‐HERC3, or HA‐HERC3‐CA as indicated. A total of 24 h after transfection, ubiquitinated proteins were pulled down using Ni‐NTA beads and the ubiquitinated YAP proteins were analyzed by using anti‐FLAG antibody.
  4. β‐TrCP1 and β‐TrCP2 were efficiently knocked down by siRNA. Total mRNA levels of β‐TrCP1 and β‐TrCP2 in MDA‐MB‐231 cells were analyzed by qRT–PCR using primers specific to the indicated target gene. Data are shown as mean ± SEM; n = 3 biological replicates. Statistical analysis was performed using two‐tailed Student's t‐test. ***P < 0.001.

Source data are available online for this figure.