Bistris nupage sds gels

The plasmid coding for the trimera was kindly provided by Prof. E. Pick (University of Tel Aviv, Israel). It codes for the Nter-p47phox (amino acids 1-286) linked to the N-ter p67phox (amino acids 1-212), and the full length Rac1 Q61L [44 (link)]. The trimera was expressed and isolated from E.coli BL21-(DE)3-plysS. Purification of trimera was performed mainly as previously described [45 (link)]. Briefly, after a first step through a nickel affinity chromatography, the protein was further purified by size exclusion chromatography; the protein was then dialyzed overnight against a phosphate buffer (100 mM NaCl and 30 mM Na₂HPO₄, pH 7.5) and stored at −80°C. The protein concentration was estimated using a NanoDrop2000 spectrophotometer (Thermo scientific, France) and the extinction coefficient of 124,000 mol^-1 L cm^-1 at 280 nm (Expasy, Protparam). The purity of all proteins were checked by migration on 10% BisTris-NuPAGE SDS gels (Invitrogen), stained with Coomassie Brilliant Blue and quantified by the ImageJ software.

HEK293 cells were transfected with either pcDNA3.1-His-3xFLAG-FAT10 [19 (link)], or with pcDNA3.1-His-3xFLAG-FAT10 ΔGG [23 (link)], by using TransIT-LTI Transfection Reagent (Mirus), or endogenous FAT10 expression was induced as recently described [23 (link),47 (link)]. To maintain constantly high levels of FAT10 until harvest after 72 h, cells were transfected a second time or retreated with proinflammatory cytokines after 48 h. After 24, 48, or 72 h of ectopic expression, cells were harvested, lysed, and cell lysates were subjected to separation on 4–12% gradient Bis/Tris NuPAGE SDS gels (Invitrogen). Western blot analysis was performed using a directly labeled peroxidase-conjugated mAb against HA-7 or a HRP-conjugated mAb against FLAG M2 (both Sigma), or a rabbit pAb against ubiquitin (DakoCytomation, Glostrup, Denmark). Antibody against β-actin (Abcam) was used as a loading control.