(A) Cytc is acetylated in castrate-resistant (PC3) and castrate-sensitive (VCaP) prostate cancer xenograft tissues compared to normal human kidney tissue and recombinant human Cytc. (B) Cytc is acetylated in cultured PC3 and VCaP cells, but not acetylated in lung and kidney cancer tissues. (C) Cytc is acetylated in human prostate cancer tissues, but not acetylated in normal prostate tissue, lung cancer tissue, and MDA-MB-231 cells. (D) Mass spectrum of Cytc peptide TGQAPGYSYTAANK(acetyl)NK identifying Cytc K53 acetylation (underlined). This spectrum represents one immunoprecipitated castrate-sensitive tumor xenograft. The acetyl moiety was unambiguously assigned to this lysine via fragment ions y4+, y5+, etc., and b14+ containing 42 extra mass units for the acetyl group. The peptide sequence was unambiguously revealed by fragment ions b3+, b4+, b11+, b14+, y4+, y5+, y7+, y8+, y9+, y10+, y11+, y12+, and y13+. (E) Coomassie blue-stained gel showing the purity of recombinant Cytc overexpressed and purified from bacteria. All 4 Cytc variants displayed a single band on a 10% tris-tricine SDS-polyacrylamide gel at around 12 kDa.