Skip to main content
. 2017 Mar 21;18(5):809–825. doi: 10.15252/embr.201643617

Figure 4. CUEDC2 enhances LDHA protein expression by protecting it from proteasomal degradation via 14‐3‐3ζ.

Figure 4

  • A
    LDHA protein levels were detected by Western blot in PLC cells expressing shCUEDC2 in the presence or absence of proteasome inhibitor MG132 (5 μmol).
  • B
    HA‐Ub, FLAG‐LDHA, and shCUEDC2 cotransfected HEK293T cells were treated with MG132 (5 μmol) before lysis. Equal amount of proteins were used for immunoprecipitation with anti‐FLAG antibody, followed by blotting with anti‐HA or anti‐LDHA.
  • C
    The expression of proteins which are predicted to bind to LDHA by bioinformatic analysis was examined by Western blot in PLC cells stably expressing shCUEDC2 or overexpressing CUEDC2.
  • D
    Lysates from PLC cells overexpressing HA‐CUEDC2 and FLAG‐14‐3‐3ζ were used for immunoprecipitation with control IgG, anti‐FLAG (left panel), or anti‐HA (right panel), followed by blotting with anti‐HA or anti‐FLAG.
  • E
    14‐3‐3ζ protein levels were detected by Western blot in PLC cells expressing shCUEDC2 in the presence or absence of MG132 (5 μmol).
  • F
    HA‐Ub, FLAG‐14‐3‐3ζ, and shCUEDC2 cotransfected HEK293T cells were treated with MG132 (5 μmol) for 6 h before lysis. Equal amount of proteins were used for immunoprecipitation with anti‐FLAG antibody, followed by blotting with anti‐HA or anti‐14‐3‐3ζ.
  • G
    Lysates from PLC cells overexpressing FLAG‐LDHA and HA‐14‐3‐3ζ were used for immunoprecipitation with control IgG, anti‐FLAG (left panel), or anti‐HA (right panel), followed by blotting with anti‐HA or anti‐FLAG.
  • H
    LDHA protein levels were analyzed by Western blot in PLC cells with silenced expression of 14‐3‐3ζ by shRNAs.
  • I
    HA‐Ub, FLAG‐LDHA, and sh14‐3‐3ζ cotransfected HEK293T cells were treated with MG132 (5 μmol) before lysis. Equal amount of proteins were used for immunoprecipitation with anti‐FLAG antibody, followed by blotting with anti‐HA or anti‐LDHA.
  • J
    LDHA protein levels were detected by Western blot in PLC cells with forced expression of CUEDC2 and sh14‐3‐3ζ.
  • K–M
    Cellular glucose uptake and lactate production (K), cell growth curve (L), and apoptosis (M) were determined in PLC cells stably overexpressing CUEDC2 with further knockdown of 14‐3‐3ζ by shRNAs.
  • N
    A diagram shows CUEDC2 accumulated 14‐3‐3ζ protein which further interacts with and stabilizes LDHA protein.
Data information: (K–M) Data are presented as mean (± SD); n = 3 in each group. *P < 0.05 as compared to EV + NTC group. P was calculated by Student's t‐test. NS: Not significant between indicated groups. β‐Actin served as loading control. Source data are available online for this figure.