Figure 2.

Glucose Regulation of IAP Proteolysis

Cells were grown to confluency in 25, 12.5, or 5 mm glucose before overnight incubation in SFM. After lysis, equal amounts of protein were separated by SDS-PAGE, and IAP was visualized by immunoblotting (IB) with an anti-IAP monoclonal antibody (B6H12). A, To visualize both intact and residual fragment, the anti-IAP monoclonal antibody (B6H12) was used (top panel). Membranes were stripped and reprobed with an anti-SHPS-1 antibody (bottom panel). The graph shows the difference in intact IAP detected with B6H12 in SMC grown in 25 mm glucose and those grown in 5 mm glucose expressed as arbitrary scanning units (mean ± sd, n = 3; *, P < 0.05). To control for nonspecific immunoprecipitation of IAP, cell lysates from SMC grown in 25 mm glucose were immunoprecipitated with either B6H12 (lane 1) or mouse control Ig (Con IgG; lane 2) and immunoblotted with B6H12. To detect IAP, samples were separated without the addition of reducing agents and thus the immunoprecipitating antibody remained intact and was detected by the antimouse horseradish peroxidase secondary antibody, and this is indicated with the arrow at the top of the blot and labeled msIgG. B, To determine whether cleavage resulted in loss of the extracellular domain, the anti-IAP polyclonal antibody (R569) raised using a peptide that contained amino acids 43–61 of IAP as an immunogen was used (top panel). To detect the fragment shed after cleavage, conditioned medium samples were concentrated, and then IAP was visualized using the anti-IAP antibody R569 (second panel). Cell lysate samples were also immunoblotted with nonimmune rabbit serum (Con IgG). The membranes were stripped and reprobed with an anti-SHPS-1 antibody (bottom panel). The graph shows the difference in intact IAP detected with R569 in SMC grown in 25 mm glucose and those grown in 5 mm glucose expressed as arbitrary scanning units (mean ± sd, n = 3; *, P < 0.05). C, Schematic representation of IAP to illustrate the regions recognized by the antibodies used to detect intact and fragmented IAP.