Immunoblotting was used to measure expression of receptor for AGEs (RAGE), and intracellular markers of autophagy (LCIII B) and NETosis (neutrophil elastase). To determine the impact of hyperglycemia on NETosis and autophagy, PMNs isolated from those with poorly controlled diabetes were left stimulated. PMNs isolated from age and sex matched healthy volunteers were incubated in the presence or absence of 5 and 15 mM glucose for 30 and 120 min. On completion of incubation, samples were processed for western blotting according to the standard protocol. Briefly, glucose treated and untreated neutrophils were lysed in 2X laemmli sample buffer, sonicated (to shear DNA) at 50% amplitude for 15 s on ice, boiled at 95°C for 10 min, and loaded on to 12% (wt/vol) polyacrylamide gels for separation of proteins. Once the 6 kDa band of protein ladder (See Blue Plus-Invitrogen) reached the dye front at the end of the gel, the gels were removed from the cast and prepared for transfer to nitrocellulose membranes. Nitrocellulose membranes containing neutrophil proteins were blocked for 1 h in PBS containing 5% non-fat milk and 0.1% (wt/vol) Tween, to prevent non-specific binding of antibodies. After blocking, blots were washed and incubated with anti–neutrophil elastase (Santa Cruz) polyclonal antibody in 1:1000 dilution or anti–RAGE (Sigma-Aldrich) in 1:1000 or anti-LCIIIB (Abcam) or anti-GAPDH (santa-cruz) in 1:1000 dilution in PBST (Phosphate Buffered Saline pH7.4 + 0.1% Tween) containing 5% non-fat milk. Bound antibody was detected with enhanced chemiluminescence using horseradish peroxidase–conjugated anti–mouse-Ig secondary antibodies (southern biotech) in a dilution of 1:1000.
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