Journal: The Journal of Biological Chemistry

Article Title: Site-specific 5-hydroxytryptophan incorporation into apolipoprotein A-I impairs cholesterol efflux activity and high-density lipoprotein biogenesis

doi: 10.1074/jbc.RA119.012092

Figure Lengend Snippet: 5-OHTrp72 apoA-I does not induce VCAM1 in primary endothelial cells. Recombinant apoA-I proteins used in cell-based VCAM1 expression assays were analyzed by Coomassie-stained denaturing gel (A) and MS (B). Arrow, apoA-I. B, an aliquot of the purified/modified proteins was digested in solution with trypsin and analyzed by nano-LC/MS/MS as described under “Experimental procedures.” The graph in B depicts the extent of oxidation at different tryptophan positions. C, surface VCAM1 protein expression was analyzed by cell-based ELISA (see “Experimental procedures”) in BAECs incubated with recombinant WT and 5-OHTrp72 apoA-I. WT apoA-I oxidized by a myeloperoxidase/hydrogen peroxide/chloride system (MPO-H2O2-Cl−) was used as the positive pro-inflammatory agonist in this assay. Serum-free media (SFM) served as the negative control. The median value for each group and batch number of recombinant apoA-I are indicated. Overall p value was determined by one-way ANOVA analysis. p values represent comparisons between native WT MPO-oxidized and WT apoA-I and were determined using Student's t test.

Article Snippet: Surface VCAM1 protein was determined using goat polyclonal anti-VCAM1 primary antibody (1 h at room temperature; 1:4000 in 0.1% casein/PBS; sc-1504, Santa Cruz Biotechnology, Inc.) with anti-goat IgG antibody conjugated with horseradish peroxidase (1 h at room temperature; 1:5000 in 0.1% casein/PBS; sc-2020, Santa Cruz Biotechnology) as a secondary antibody, detection by SureBlue tetramethylbenzidine peroxidase substrate (KPL Inc., Gaithersburg, MD), and measurement of absorbance at 450 nm on a 96-well plate reader (Spectramax 384 Plus, Molecular Devices, Sunnyvale, CA) after the addition of 1 m HCl to stop the reaction.

Techniques: Recombinant, Expressing, Staining, Purification, Modification, Liquid Chromatography with Mass Spectroscopy, In-Cell ELISA, Incubation, Negative Control