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. 2018 Apr 17;9(29):20323–20338. doi: 10.18632/oncotarget.24859

Figure 2.

Figure 2

(A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.