Hrp conjugated mab against flag m2

The in vitro reaction buffer contained 20 mM Tris/HCL (pH 7.6), 50 mM NaCl, 10 mM MgCl₂, 4 mM ATP, 0.1 mM dithiothreitol, 5 U·mL^-1 inorganic pyrophosphatase, 20 mM creatine phosphate, 4 μg·mL^-1 creatine phosphokinase (all from Sigma, Buchs, Switzerland), supplemented with 1x protease inhibitor mix (Roche, Rotkreuz, Switzerland). Recombinant proteins (S1 Methods) were added to a final volume of 40 μl of 1x reaction buffer in the following amounts: FLAG-UBA6, 1 μg; 6His-USE1, 6 μg; FAT10, 4 μg; ubiquitin, 25 μg; UBE1, 10 μg. The reaction mixture was incubated at 30°C for 60 min with shaking and 5x gel sample buffer with or without 4% 2-mercaptoethanol (2-ME) was added, where indicated. The proteins were separated on 3–8% gradient Tris/Acetate NuPAGE SDS gels (Invitrogen, Lucerne, Switzerland), and subjected to western blot analysis with a rabbit polyclonal antibody against huFAT10 [4 (link)], a rabbit pAb against UBE1 (Enzo Lifesciences, Lausen, Switzerland), a directly peroxidase-conjugated mAb against 6-His, a HRP-conjugated mAb against FLAG M2 (both from Sigma, Buchs, Switzerland), or a pAb against ubiquitin (DakoCytomation, Hamburg, Germany).

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.