The IHC staining pattern of E2F4 was evaluated. Briefly, after incubation with the E2F4 primary antibody (dilution 1:300, [EPR8259] (ab150360) Abcam, Cambridge, MA), the tissue microarray was probed with biotinylated goat anti-rabbit secondary antibody rabbit IgG, (Dilution: 1:10,000, Cell Signaling Technologies, Inc.), and the remainder of the process was carried out according to a previously published study 27.

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