Table 1.

Combination of approaches to characterize vimentin lipoxidation and assessment of consequences of vimentin lipoxidation.

Activity	Approach	Reference
Identification and Characterization of Vimentin
Identification as putative target for lipoxidation	15d-PGJ2B→Electrophoresis → WB → Detection of biotin → MALDI-TOF-MS	Stamatakis et al. [284]
	HNE → Electrophoresis → Anti-HNE-WB → Nano-LC-MALDI-MS/MS	Chavez et al. [283]
	Cell Senescence→Electrophoresis → Anti-HNE-WB → MALDI-TOF-MS	Aldini et al. [220]
Confirmation of lipoxidation site in cell	cyPG(biotinylated) → Immunoprecipitation → Detection Biotin	Aldini et al. [220]
	cyPG(biotinylation) → Immunoprecipitation → SDSPAGE → LCMS/MS	Gharbi S et al. [282]
	Carbonyl derivatization → Avidin enrichment → MS/MS	Chavez et al. [283]
Validation of lipoxidation site (Cys328)	Transfection → cyPG(biotinylation) → Avidin enrichment → SDSPAGE → WB	Gharbi et al. [275]
	Carbonyl derivatization → Avidin enrichment → MS/MS	Chavez et al. [283]
Analysis of lipoxidation in vitro	cyPG(biotinylation) → SDSPAGE → Biotin Detection	Gharbi et al. [282]
	cyPG(biotinylation)/HNE → SDSPAGE → Biotin Detection/Anti-HNEWB	Aldini et al. [220]
Confirmation of lipoxidation site in vitro (Cys328)	cyPG(biotinylation) → Digestion and Avidin enrichment → MALDI-TOF-TOF MS/MS	Gharbi et al. [282]
	In vitro competition assays	Aldini et al. [220]
Functional Assessment of Vimentin Lipoxidation
In vitro assessment of lipoxidation: Filaments derangement	HNE → Polymerization (NaCl Induced) → Filaments electron microscopy	Aldini et al. [220]
In cell assessment of lipoxidation (Condensation of network by HNE treatment)	Transfection of cells (expressing vimentin) with GFP → HNE treatment → Confocal microscopy	Aldini et al. [220]
Cys328 in function consequences of vimentin lipoxidation: network condensation and filaments preservation in vimentin Cys328ser	Transfection of vimentin or Cys328ser in cell (vimentin deficient) → HNE treatment → Immuno-fluorescence → Confocal microscopy	Aldini et al. [220]