Sc 30089

For the analyses of EL in heparin media 40 µL of heparin media were supplemented with 6 x loading buffer [20% (w/v) glycerol, 5% (w/v) SDS, 0.15% (w/v) bromophenol blue, 63 mmol/L Tris-HCl, pH6.8 and 5% (v/v) ß-mercaptoethanol], and boiled for 10 min before loading. Samples were analyzed by SDS-PAGE (10% gel) and with subsequent immunoblotting using EL-specific antibody exactly as described^{10 (link)}. Mouse apoA-I content was analyzed in aliquots of serum (1.5 µL), apoB-DS (2 µL) and FPLC fractions (10 µL). Samples were supplemented with 6 x loading buffer, boiled for 10 min and electrophoresed on 12% SDS-PAGE. After blotting the membranes were incubated at 4 °C overnight with mouse anti-apoA-I antibody (Santa Cruz Biotechnology, sc-30089, LOT D2913, dilution 1:500, Heidelberg, Germany). Following washing and incubation with appropriate secondary antibody (Dako, Vienna, Austria), protein signals were visualized by incubation with Millipore Western Blotting Substrate (Millipore Corporation, Billerica, USA) using ChemiDoc system (Bio-Rad Laboratories, Vienna, Austria).

HDL-associated proteins in isolated HDL (10 µg), FPLC fractions (10 µL), serum (1.5 µL), or apoB-DS (2 µL) were quantified using antibodies specific for human/mouse PON1 (abcam-ab24261, dilution 1:1000, Cambridge, UK), human apoA-I (Novus biological, NB100-65491, dilution 1:3000, Littleton, CO, USA), or mouse apoA-I (Santa Cruz Biotechnology, sc-30089, LOT D2913, dilution 1:500, Heidelberg, Germany) as described [27 (link),30 (link)].

Western blotting was carried out essentially as described previously [23 ]. A total of 30 μg of protein were separated by 12% SDS-PAGE and proteins subsequently were transferred to 0.2 μM nitrocellulose membranes by a wet transfer system. The membranes were blocked with 5% nonfat skim milk in 1x TBS for 1 hr. After blocking, the membranes were incubated with either a 1 : 3000 dilution of a mouse monoclonal anti-human haptoglobin antibody (sc-365396, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 hrs and subsequently incubated with a 1 : 8000 dilution of a rabbit anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (A9044, Sigma, Sigma-Aldrich, St Louis, MO) for 1 hr or with a 1 : 8000 dilution of a polyclonal rabbit anti-human apolipoprotein A-I antibody (sc-30089, Santa Cruz Biotechnology Inc.) for 2 hrs followed by a 1 : 4000 dilution of a HRP-conjugated goat anti-rabbit IgG polyclonal antibody (Pierce Biotechnology, USA) for 1 hr. Protein expression signal was developed using the Enhanced Chemiluminescence Plus system (Amersham Biosciences).