Fig. 4.

Composite figure of western blots demonstrating secretion of different complement proteins into conditioned media by unstimulated (lane 1), and Aβ (lane 2), IFN-γ (lane 3) and IL-1β (lane 4) stimulated HCSMC. Diluted human serum (lane 5) was used as a positive control. Conditioned media samples from HCSMC cultures were concentrated with Strataclean resin; the bound proteins were eluted from the resin, separated through polyacrylamide gels and transferred to nitrocellulose membranes. Replicate membranes were probed with the indicated antibodies. Polypeptide(s) with the expected molecular weights were detected in each sample except for C1q and C5. The high molecular weight bands in C4 antibody probed HCSMC samples represent the unprocessed form of complement C4. As a surrogate loading control, in order to demonstrate the reliability and reproducibility of the Strataclean concentration method, replicate aliquots of samples containing the same amount of GFAP protein were processed and detected in parallel (GFAP loading).