Lyophilized bovine EP (bEP) was reconstituted to a concentration of 0.5 mg/ml and stored in aliquots at ?80��C until further usage. The final concentration of bEP was 0.05 ��g/ml and the activity was determined as described for human EP. 1,10-phenanthroline (used to stop the activation of human recombinant EP) had no effect on the inhibition of bovine EP by PCI (data not shown). SDS-PAGE selleck chem and Western Blotting SDS-PAGE was performed according to the method of Laemmli [56] using 10% acrylamide gels. EP (60 nM) and PCI (440 nM) were preincubated in 10 ��l TCNB buffer for varying times at 37��C. The reactions were stopped by the addition of an equal volume of Laemmli buffer (125 mM Tris, 20% glycerol, 4.1% SDS, 0.01% bromphenol blue with or without 10% 2-mercaptoethanol) and heating the samples for 5 min at 95��C.
For the zero-time point the proteins were added directly into Laemmli buffer. Thereafter, they were applied to a 10% acrylamide gel. After electrophoresis, the gels were transferred to PVDF membranes for Western blotting. Blocking of membranes was done with 5% dry milk in PBST (135 nM NaCl, 1.3 mM KCl, 2.5 mM Na2HPO4, 0.5 mM KH2PO4, 0.1% Tween-20) either at 4��C overnight or at room temperature for 1 h. Incubations with the primary antibody were also done in 5% milk in PBST at 4��C overnight or at room temperature for 1 h. The membranes were washed 5 times (5 min each) in PBST. The secondary peroxidase-labeled antibody was applied for 1 h in PBST with 5% dry milk. After another washing step, the peroxidase reaction was detected with SuperSignal West Femto.
Primary antibodies used in Western blotting were monoclonal mouse anti-serpinA5 IgG (0.5 ��g/ml), monoclonal anti-PRSS7 (enteropeptidase) IgG (1 ��g/ml) and polyclonal rabbit anti-EP-light-chain IgG (78 ��g/ml). The respective secondary antibodies were HRP-conjugated donkey anti-rabbit IgG (0.11 ��g/ml), and HRP-conjugated sheep anti-mouse IgG (0.13 ��g/ml). Acknowledgments We would like to thank Christian Sch?fer (Medical University of Vienna, Austria) for helpful discussions and Bettina Sarg (Innsbruck Medical University, Austria) for analyzing bovine EP by Edman degradation. We also want to thank J. Evan Sadler and Lisa Westfield (Washington University School of Medicine, St. Louis, MO) for providing EP light chain antibodies. Footnotes Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the FWF-Austrian Science Fund grant P20248-B09 to MG, grant P22160-B09 to MG, and grant P20386-B09 to MF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Gastrointestinal neuroendocrine tumors (GI-NETs) by tradition are known as carcinoids and AV-951 they are rare tumors.