Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

Figure 3

Separation by two-dimensional electrophoresis (2-DE) of the proteome of HL-60 cells under basal conditions ((a), (b)) and after exposure to 10 μM HNE at 37°C for 2 h ((c), (d)). Total cell extracts, each containing from 50 to 200 μg of total protein, were prepared from 1 × 10⁶ HL-60 cell aliquots. The gels in (a) and (c) were silver-stained, and those in (b) and (d) were electrophoretically transferred to PVDF membranes and probed with a murine anti-HNE-histidine monoclonal antibody, which was detected with a secondary anti-murine IgG antibody, with chemiluminescent technique. Molecular weight standards (MW St) in kDa are marked at right of the gels in (a) and (c). The signal detected by the use of the anti-HNE-histidine antibody in the immunoblots of (b) and (d) was used to identify, by visual alignment, the corresponding spots in the gels shown in (a) and (c). The spots thus identified were assigned numbers. The spots identified in the proteomes of both control and test cells are circled in purple, and those selectively identified in HNE-treated cells are circled in red.

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