Journal: Cancer Communications
Article Title: Proliferation and Apoptosis Adaptor Protein 15 (PEA15), a Potential Oncogenic Regulator of VHL and HIF1A Identified through Proteomic Analysis in Hepatocellular Carcinoma
doi: 10.34133/cancomm.0020
Figure Lengend Snippet: The N-terminal helices of proliferation and apoptosis adaptor protein 15 (PEA15) mediate von Hippel–Lindau tumor suppressor (VHL) binding and promote hypoxia-inducible factor 1A (HIF1A) stabilization and hepatocellular carcinoma (HCC) cell proliferation. (A) Results of a glutathione S -transferase (GST)–PEA15 pull-down assay with purified GST-fused PEA15 and recombinant VHL. Recombinant His-tagged VHL (His-VHL) was incubated with purified GST-fused fragmented PEA15 proteins as indicated, and a precipitated protein complex with Glutathione-Sepharose 4B beads was immunoblotted with anti-His probe antibodies. The H numbers in fragmented PEA15 indicate α-helix numbers in N-terminal regions of PEA15. The numbers in parentheses (except that for the input) indicate the residue numbers in PEA15. #PEA15 fragments that can interact with VHL. (B) Cell viability assay results for HepG2 cells with overexpression of fragmented PEA15. Deletion of the N-terminal region of PEA15 significantly abrogated the proliferation-promoting activity of PEA15 in these cells. Expression of fragmented PEA15 did not increase the stability of HIF1A. The whiskers indicate SE. (C) Cell viability assay results for HepG2 cells with overexpression of the N-terminal region of PEA15. The first 4 helices (H1–4) of PEA15 are sufficient to promote the proliferation of HepG2. Expression of PEA15-H1–4 increases the stability of HIF1A. The whiskers indicate SE. *** P < 0.001 (Student’s t test). His-VHL, histidine-tagged VHL; FL, full length; NT, N-terminal; CT, C-terminal; H, helix; PEA15 FL-OE, PEA15 full-length overexpression; PEA15ΔH1–2-OE, helix 1–2 deleted PEA15 overexpression; PEA15ΔH1–4-OE, helix 1–4 deleted PEA15 overexpression; PEA15H1–4-OE, PEA15 helix 1–4 fragment overexpression; GST-PEA15, GST-fused PEA15; PEA15-Flag, Flag-tagged PEA15.
Article Snippet: For the GST-PEA15 pull-down assay, 1 to 5 μg of GST-PEA15 proteins were incubated with the same amount of recombinant His-VHL (TP760451, OriGene Technologies) in binding buffer (50 mM tris–HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF) at 4 °C for 4 h to overnight.
Techniques: Binding Assay, Pull Down Assay, Purification, Recombinant, Incubation, Residue, Viability Assay, Over Expression, Activity Assay, Expressing
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