Figure 4.

CCEU covalently alkylates PHB on an Asp residue. (a) 2D gel analysis of proteins extracted from cells treated for 24 h with 100 μM CCEU (bottom) revealed one additional spot (P2) when compared to DMSO-treated cells (top). (b) Western blot analysis showed that two PHB spots were only present in CCEU-treated B16 cells, the basic one being labelled by [¹⁴C]-CCEU as demonstrated by autoradiography of the membrane. (c) MALDI-TOF-MS comparison of the two proteins identified as PHB. The ion at m/z 3125 corresponds to the peptide [Gly 44-Arg 70] containing the only Cys residue of PHB for the acidic PHB (top) or basic PHB (bottom). (d) MALDI-TOF-MS comparison of the PHB spots. An ion at m/z 720.41, corresponding to peptide [Ala 36-Arg 41] present in the original acidic PHB (top) was absent in the basic PHB, which in contrast contained an ion at m/z 964.56 (bottom). The mass difference (Δm=244.15) was in accordance with the presence of a CCEU residue on peptide [Ala 36-Arg 41]. ^* Corresponds to trypsin autolysis peptide (m/z=842.5100). CCEU, cyclohexylphenyl-chloroethyl urea; DMSO, dimethylsulphoxide; MALDI-TOF-MS, matrix-assisted laser desorption/ionisation–time of flight–mass spectrometry; PHB, prohibitin.