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 6

Immunoprecipitation and western blot analysis of HSP60 expression and of HNE-HSP60 adduct formation in HL-60 and THP-1 cells treated with HNE or with native or modified LDL. (a) left: lanes 1 and 3, control HL-60 cells incubated without additions for 2 and 6 h, respectively; lanes 2 and 4, HL-60 cells exposed to 10 μM HNE for 2 and 6 h, respectively. β-actin was used as a quantitative reference. (b) left: immunoprecipitation of HL-60 cell protein extracts with anti-HNE-histidine antibodies (aHNE), followed by western blot with anti-HSP60 antibodies (aHSP60) (above); immunoprecipitation of HL-60 cell protein extracts with anti-HSP60 antibodies (aHSP60), followed by western blot with anti-HNE-histidine antibodies (aHNE) (below). (c) left: western blot with anti-HNE-histidine antibodies of the protein extracts subjected to immunoprecipitation with N-20 anti-HSP60 antibodies, from THP-1 cells differentiated with PMA and exposed for 4 h to 10 μM HNE (HNE), 20 μg/mL of native LDL (LDL), 20 μg/mL of LDL modified with 1 mM HNE (HNE-LDL) at 37°C for 20 h, or 15 μg/mL of oxLDL(Cu), that is, LDL modified by metal-catalyzed oxidation with 20 μM Cu(SO₄) at 37°C for 20 h. The positions of the molecular weight standards (MWSt) of 50 and 75 kDa are marked at left. All panels, at right: densitometric scans, in the form of histograms, of the HSP60- and HNE-immunoreactive bands of the respective immunoblots at the left side of the panels.

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