Protein truncation and dephosphorylation events detected by 2DE in DNR-treated HL60 cells. (A,B) The boxed area shows the position (A) and detail (B) of the methylosome subunit chloride conductance regulatory protein (pICln) in Sypro Ruby-stained 2DE gels (pI 3-10) with extract of control HL60 cells. (C) Like panel B, except that boxed area is from gel with extract from HL60 cells treated for 6 hours with 8 μmol/L DNR. (D) Immunoblotting of pICLn on extracts of HL60 cells treated with 1.6 μmol/L DNR for the times indicated. Note truncation of pICLn from 29 to 27 kDa, starting after 3 hours of DNR exposure. (E) The spots circled with solid lines show the MS-detected ribosomal protein P2 (rpP2) and eEF1Ba (25 kDa). (F) After 6 hours with 8 μmol/L DNR, a significant proportion of the eEF1Ba appeared as a 17 kDa form (arrow), whereas rpP2 appeared as 3 spots of similar size but with different pI. (G and H) MALDI analysis of the 25-kDa (G) and 17-kDa (H) variants of eEF1Ba revealed a tryptic peptide (1732.87), present only in the 17-kDa ΔeEF1Ba. It was identified by tandem mass spectrometry to have a predicted caspase cleavage site (DETD) at its C terminus. (I-J) The relative distribution of eEF1Ba (circled) and ΔeEF1Ba (arrow) in 2DE gels (pI 4-5) of extract from blasts from patients with AML (M2, patient 5) treated in vitro for 6 hours with vehicle (I) or 8 μmol/L DNR (J). The gels were silver-stained.