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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Cancer Res. 2018 Mar 9;78(11):2897–2910. doi: 10.1158/0008-5472.CAN-17-3531

Figure 1. Identification of Peli1 as a bona fide Mdmx interacting protein in cancer cells.

Figure 1

(A) Silver staining of affinity-purified Mdmx complexes from the cell extracts of the FH-Mdmx A375 stable cells and the parental A375 cells. Mdmx-interacting partners were identified by mass spectrometry, and the Peli1 peptide sequences are presented.

(B) Co-immunoprecipitation of FH-Peli1 with Myc-Mdmx from H1299 cells. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Mdmx alone or with Myc-Mdmx and FH-Peli1 together were subjected to western blot with anti-Myc (top) and anti-HA antibody (lower).

(C) Co-immunoprecipitation of FH-Mdmx with Myc-Peli1 from H1299 cells. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Peli1 alone or with Myc-Peli1 and FH-Mdmx together were subjected to western blot with anti-HA (top) and anti-Myc antibody (lower).

(D) Direct interactions of GST-Peli1 with Mdmx. The GST-Peli1 fusion protein and GST alone were used in a GST pull-down assay with the FH-Mdmx protein purified from H1299 cells. Elutes from GST beads were subjected to western blot with anti-HA antibody (top) after Ponceau S staining (lower). Asterisk indicates GST-Peli1 fusion protein.

(E) Direct interactions of GST-Mdmx with Peli1. The GST-Mdmx fusion protein and GST alone were used in a GST pull-down assay with the FH-Peli1 protein purified from H1299 cells. Elutes from GST beads were subjected to western blot with anti-HA antibody (top) after Ponceau S staining (lower). Asterisk indicates GST-Mdmx fusion protein.

(F) Co-immunoprecipitation of endogenous Mdmx with Peli1 from A375 melanoma cells. Western blot analysis of whole cell extract (lane 1) and immunoprecipitates with a control IgG (lane 2) or a Peli1-specific antibody (lane 3) by anti-Peli1 monoclonal antibody (top) or anti-Mdmx polyclonal antibody (lower).

(G) Co-immunoprecipitation of endogenous Peli1 with Mdmx from A375 melanoma cells. Western blot analysis of whole cell extract (lane 1) or immunoprecipitates with control IgG (lane 2) or anti-Mdmx polyclonal antibody (lane 3) by anti-Mdmx polyclonal antibody (top) or anti-Peli1 monoclonal antibody (lower).