Journal: PLoS ONE
Article Title: Protoporphyrins Enhance Oligomerization and Enzymatic Activity of HtrA1 Serine Protease
doi: 10.1371/journal.pone.0115362
Figure Lengend Snippet: ( A ) MPPs displayed differential activity in affecting The pattern of extracellular HtrA1 complexes (arrowheads) from HeLa cell low-serum conditioned medium is differentially altered by treatment with MPPs (25 mM, 37°C, 1 hr), as demonstrated when probed with polyclonal anti-HtrA1 antibody. ( B ) MPPs may induce conformational changes upon binding to HtrA1. The accessibility of N-terminal and C-terminal epitopes in the presence of MPPs was determined by ELISA and compared to DMSO controls. Error bars indicate the standard deviation. ( C ) Extracellular HtrA1 (arrows) could be precipitated from HEK-HtrA1 conditioned medium using HEMIN-conjugated agarose beads but not control, unconjugated agarose beads. ( D ) Competitive binding experiments using conditioned medium from HEK-HtrA1 or HeLa cells pre-incubated with MPPs revealed reduced recovery of HtrA1 (arrows) in the presence of competitor compounds when probed with polyclonal anti-HtrA1 antibody. ( E ) Degradation of Fibulin 5 in fixed HeLa cells treated with HtrA1 conditioned medium was enhanced in the presence of TPP, ZPP and PPP-IX. RMA: Rosmarinic acid, TPP: tin protoporphyrin IX, ZPP: zinc protoporphyrin IX, PPP-IX: protoporphyrin IX, HEM: HEMIN, CPP: cobalt protoporphyrin IX, CM: conditioned medium.
Article Snippet: All antibodies were purchased from Cell Signaling Technology except monoclonal anti-HtrA1 and polyclonal anti-HtrA2 (R&D Systems), rabbit polyclonal anti-Fibulin-5 (Millipore), monoclonal anti-V5 (Invitrogen), and rabbit polyclonal anti-HtrA1 (kind gift from Dr. Sascha Fauser, University of Cologne ).
Techniques: Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Incubation