Myc 9e10 antibody

Hek293T cells were transiently transfected with the myc-tagged PHMA Trib2 or PHMA control vectors as previously described^{15 (link)}. The transfected cells were harvested at 24 h and whole cell lysates were prepared using ice-cold Hepes buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5% glycerol, with protease and phosphatase inhibitors). Crosslinking was performed using dithiobis-succinimidylpropionate (DSP) (Thermo Fisher Scientific) at a concentration of 1.5 mM, and the reaction quenched using Tris (pH 7.4) (50 mM). One milligram of precleared lysates were incubated with Myc9E10 antibody (Santa Cruz Biotechnology) or normal mouse IgG overnight at 4 °C, followed by incubation with Protein A/G UltraLink™ Resin (Thermo Fisher Scientific) for 1.5 h at 4 °C. The samples were washed in Tris buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5% glycerol, with protease and phosphatase inhibitors), eluted in Laemmli buffer and analysed by western blotting.

The SNX8 antibody was purchased from Novus. The Aβ (6E10) antibody was purchased from Biolegend. GAPDH, GFAP, and β-actin antibodies were purchased from Cell Signaling Technology. NeuN and Giantin antibodies were purchased from Abcam. The Myc (9E10) antibody was purchased from Santa Cruz Biotechnology. The Iba1 antibody was purchased from Wako. The tau PHF1 antibody, Alexa Fluor 594 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 635 goat anti-mouse IgG, goat anti-rabbit IgG (H + L) secondary antibody HRP, and goat anti-mouse IgG (H + L) secondary antibody HRP were purchased from Thermo Fisher Scientific. Antibodies against APP (369) and PS1-NTF (Ab14) were generated in-house (Thinakaran et al., 1996 (link); Xu et al., 1997 (link)). The sAPPα (B436) antibody has been described previously (Eggert et al., 2009 (link)).