Denaturing loading buffer

10% cattle brain homogenates were mixed with denaturing loading buffer (Invitrogen), heated for 10 min at 95 °C and fractionated in 4–12% NuPAGE gels (Invitrogen). Proteins were transferred to a nitrocellulose membrane (GE Healthcare), blocked with 10% milk, and incubated with rabbit anti-Aβ42 polyclonal antibody (Covance). After incubation with secondary antibody, Aβ42 was visualized by chemoluminescence using ECL plus (GE Healthcare) in a dark chamber (BioRad).

HEK-293 T cells (kindly provided by Dr. G. Caignard) were plated in 6-well plates (2 × 10⁶ cells per well) and 24 h later transfected with 500 ng of GST-tagged vectors and 300 ng 3xFlag-tagged vectors. Two days after transfection, HEK-293 T cells were washed in phosphate-buffered saline (PBS), and then resuspended in lysis buffer (20 mM MOPS-KOH pH 7.4, 120 mM of KCl, 0.5% Igepal, 2 mM β-Mercaptoethanol), supplemented with Complete Protease Inhibitor Cocktail (Roche). Cell lysates were incubated on ice for 20 min, and then clarified by centrifugation at 15 000 g for 15 min. For pull-down analysis, 300 µl of each protein extraction was incubated for 2 h at 4° C with 35 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. Beads were washed 3 times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (Invitrogen).

To perform co-affinity purification experiments, ORFs encoding NS4 and WTAP or fragments, or CCHCR1 were transferred from pDONR207 to either pDEST27 (Invitrogen) or pCI-neo-3xFLAG expression vector to achieve GST and 3xFLAG fusion [39 (link)], respectively. HEK-293T cells were dispensed in each well of a 6-well plate (2 × 10⁶ cells), and 24 h later transfected (JetPRIME; Polyplus) with 500 ng of each plasmid DNA per well. Two days post-transfection, cells were collected in PBS and then incubated on ice in lysis buffer (20 mM MOPS-KOH pH 7.4, 120 mM of KCl, 0.5% Igepal, 2 mM β-Mercaptoethanol, supplemented with Complete Protease Inhibitor Cocktail (Roche)) for 20 min. Cell lysates were clarified by centrifugation at 14,000× g for 30 min. For pull-down analysis, protein extracts were incubated for 2 h at 4 °C on a spinning wheel with 30 μL of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. Beads were then washed 3 times for 5 min with ice-cold lysis buffer and on a spinning wheel and, proteins were recovered by boiling in denaturing loading buffer (Invitrogen).