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. 2016 May 4;17(6):874–886. doi: 10.15252/embr.201541685

Figure 4. HUWE1 interacts with PCNA .

Figure 4

  • A
    Schematic representation of full‐length HUWE1. Shown is the PIP‐box domain we describe here (aa 3880‐3887). ARLD: Armadillo repeat‐like domain; UBA: ubiquitin‐associated domain; BH3: Bcl2‐homology three domain; HECT: homologous to the E6‐AP carboxyl terminus (catalytic ubiquitin ligase domain). The span of the HUWE1C‐ter (3875‐end) fragment used in subsequent studies is also indicated.
  • B–D
    Interaction between endogenous HUWE1 and PCNA in 293T cells. (B) Anti‐PCNA immunoprecipitation, showing that HUWE1 co‐precipitates. (C) Reciprocal experiment showing that PCNA co‐precipitates with HUWE1 in anti‐HUWE1 immunoprecipitation. (D) As control, endogenous HUWE1 was depleted by siRNA treatment, and extracts were subjected to anti‐HUWE1 immunoprecipitation. The HUWE1 blot shows that much less HUWE1 is precipitated by anti‐HUWE1 antibodies from HUWE1‐knockdown cells than control, as expected. PCNA is co‐precipitated by anti‐HUWE1 antibodies from control, but not HUWE1‐depleted cells, showing that the interaction is specific and not due to unspecific binding of PCNA to anti‐HUWE1 antibodies.
  • E, F
    HUWE1 co‐precipitates with PCNA in anti‐PCNA immunoprecipitation from extracts of MCF7 (E) and 8988T (F) cells, showing that the HUWE1–PCNA interaction can be detected in different cell lines.
  • G
    Co‐immunoprecipitation experiment from control and Rad18‐knockout 293T cells, showing that the HUWE1–PCNA interaction is not affected by loss of PCNA ubiquitination. Rad18‐knockout cells were obtained by CRISPR/Cas9 technology. The Rad18 and PCNA blots show loss of Rad18 and of PCNA ubiquitination, respectively.
  • H
    Myc‐HUWE1C‐ter fragment was transfected in 293T cells. Anti‐PCNA immunoprecipitation shows that the C‐terminal HUWE1 fragment interacts with PCNA.
  • I
    Coomassie staining of recombinant GST‐PCNA expressed and purified from bacteria.
  • J
    GST‐pull downs using GST‐PCNA or GST‐empty as control, and extracts of 293T cells transfected with Myc‐HUWE1C‐ter, showing interaction of this fragment with recombinant PCNA.