IFN[alpha]2-T[alpha]1 gene was directly inserted in to pET SUMO vector (Invitrogen) following the instructions of the manufacturer (Champion(tm) pET SUMO Protein Expression System, Invitrogen) using T4 DNA ligase.
Briefly, we cloned IFN[alpha]2-T[alpha]1 gene in pET SUMO expression vector (Invitrogen) downstream of SUMO gene by following instructions given in manual (Champion(tm) pET SUMO Protein Expression System, Invitrogen) with His6-tag at N-terminal end of SUMO tag for purification by immobilized metal ions chromatography.
The researchers first used an automated process to examine whether the compounds prevented SENP2 from severing the connection between a tiny metal bead and an artificial SUMO protein
created in the lab of Wei Yang, Ph.D., the study's other senior author and an associate professor at Duke University in Durham, NC.
Our previous study demonstrated that high glucose obviously induced the expression of SUMO isoforms (SUMO1, SUMO2/3) in GMCs, subsequently stimulated degradation of I[kappa]B[alpha], and triggered NF-[kappa]B signaling by weakening the interaction between SUMO protein and I[kappa]B[alpha] while promoting ubiquitination of I[kappa]B[alpha] .
Based on our findings, we suggest a new model for the activation of NF-[kappa]B inflammatory signaling: exposure of the GMCs to high glucose results in the overexpression of the SUMO E3 ligase PIASy and SUMO proteins, which causes the SUMO proteins to bind to IKK[gamma], mediating the phosphorylation and sumoylation of IKK[gamma]; the subsequent degradation of I[kappa]B[alpha] and activation of NF-[kappa]B in turn result in the processing of MCP-1 and IL-6 release from GMCs, eventually promoting the renal low-grade inflammation.
We excluded the possibility of a reaction with SUMO protein
by testing with the control protein.
The SUMO proteins
are small; most are around 100 amino acids in length and 12 kDa in mass.
These key SUMO proteins
produce subtle responses to the brain's activity levels to regulate the amount of information transmitted by kainate receptors - responsible for communication between nerve cells and whose activation can lead to epileptic seizures and nerve cell death.