Mouse anti his antibody

Equal amounts of protein (500 μg) extracted from the brains of WT and MsrA KO mice were incubated with recombinant Nedd8 (FHN8) conjugates (8 µg) in PBS at 37 °C. After the designated time of incubation, equal volumes of 2× sample buffer were added and immediately heated at 100 °C for 5 min to terminate the reaction. The reaction solution of different time points was subjected to Western blot using mouse anti-His antibody (Qiagen, Hilden, Germany) as the primary antibody. The neddylation levels of the lowest molecular mass of the His-detected Nedd8 conjugates (L.M. Nedd8) were quantified by NIH Image-J program.

For each assay, 0.5 × 10⁶ HBEC-5i cells were incubated with 100 µg/ml of IT4-VAR19 recombinant proteins (VAR19-NTS-DBLγ6 and VAR19-CIDRα1.1) diluted in PBS 0.2 % BSA. After 30 min of incubation on ice, the cell suspension was washed twice with PBS 0.2 % BSA and incubated for 30 min with a mouse anti-His antibody (QIAGEN), at 2 μg/ml in PBS 0.2 % BSA. The cell suspension was washed twice and incubated for 30 min with PE-conjugated goat anti-mouse IgG (Beckman Coulter, diluted 1:100 in PBS 0.2 % BSA). Cells were fixed overnight with PFA 4 % and washed twice with PBS. Data acquisition was performed using a BD FACScanto II flow cytometer (Becton–Dickinson, San Jose, CA, USA) and data were analysed using the FLOWJO 8.1 software (Tree Star Inc).