Proteosilver plus silver staining kit

Coding sequence for full‐length VEGI‐251 was amplified by reverse transcription‐polymerase chain reaction (RT‐PCR) by using HUVEC (human umbilical vein endothelial cell) mRNA as the template. The primer sequences as shown in supplemental Table S1 included the restriction sites Kpn I and Bam HI for PCR‐mediated cloning into the eukaryotic expression vector pEF‐BOS (Invitrogen, Carlsbad, CA). A 6× His‐Tag sequence was introduced into the protein sequence at the carboxy‐terminus. To obtain the VEGI‐251‐His protein, the expression and purification of rhVEGI‐251 protein were performed in accordance with an established method as previously described⁸, ⁹ via Ni‐NTA affinity chromatography (The QIAexpressionist, Qiagen, Chatsworth, CA, USA). The vehicle control was the product extracted by expression of the empty pEF‐BOS vector without VEGI‐251 using the same expression and purification protocol as described above. After the purified rhVEGI‐251, protein was separated by SDS‐PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and assessed with silver staining using a ProteoSilver Plus Silver Staining Kit (Sigma‐Aldrich, St. Louis, MO, USA), and then, the clear single target band was excised from the gel and subjected to mass spectrometry analysis.

Whole blood was collected via cardiac puncture and placed in BD microtainer serum separator tubes (VWR). Blood samples were then centrifuged for 5 minutes at 2000 x g to separate serum. Idexx Laboratories, Inc. (North Grafton, MA) performed serum chemistry analysis.
Two μL of urine was denatured and run on a SDS-PAGE gel and proteins were visualized using ProteoSilver Plus Silver Staining Kit (Sigma).