Validation of proteomic results. (A) A 2-DE image of the β-actin region for NB4 cultured in a 99% isotopic abundance of Leu-d3. The plots represent tryptic peptides of β-actin, heat shock protein 70, and stress-70 protein, which contain 1 leucine amino acid. The peaks of 896.0, 682.9, and 897.5 are the monoisotopic peaks for the peptides yielding the Leu-d3 amino acid, and in each plot the asterisk represents the nondeuterated form of the peptide. (B) SDS-PAGE separation of a 1:1 mixture of nontreated versus ATRA-treated NB4 at 24 hours. The ATRA-treated cells were grown in a 99% isotopic abundance of Leu-d3 media and mixed 1:1 with nontreated cells grown in normal culture media in accordance with the AACT methodology. The plots on the right represent the AACT-based quantification of tryptic peptides obtained from in-gel digests. Relative intensity comparisons of the same leucine containing peptides from nontreated and ATRA-treated NB4 yield the up-regulation of myosin heavy chain (650.7 versus 655.7), unchanged β-actin (895.9 versus 897.4), and down-regulated cofilin (669.3 versus 670.8). (C) Western blot analysis of 14-3-3ϵ, 14-3-3ζ, hnRNP K, eIF4G, Rho GDI-1, DAP5/p97/NAT1, and PML-RARα as a function of ATRA treatment, using 50 μg total protein. Antibodies to 14-3-3ϵ, 14-3-3ζ, hnRNP K, eIF4G, Rho GDI-1, DAP5/p97/NAT1, and PML-RARα were used to detect expression of specific proteins. (D) HL-60 Western blot analysis of 14-3-3ζ, hnRNP K, eIF4G, and eIF5 as a function of ATRA treatment, using 50 μg total HL-60 protein.