Rabbit anti α tubulin polyclonal antibody

Total protein from cells and tissues were extracted with RIPA buffer containing protease inhibitor cocktail and EDTA. Proteins were electrophoresed and transferred to membranes as described previously^{36 (link)}. The membranes were then blocked with 5% Blotting-Grade Blocker (Bio-Rad) in TBS-T (50 mM Tris, 138 mM NaCl, pH 7.4, 0.1% Tween-20) and subsequently incubated at 4 °C overnight with rabbit anti-ADAM10 polyclonal antibody (LifeSpan BioSciences,) or rabbit anti-α-tubulin polyclonal antibody (Cell Signaling Technology) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad).

An amount of 30 μg of total protein from the lysates of the transfected HEK293 cells was electrophoresed on 12% SDS-PAGE gels and subsequently transferred onto nitrocellulose membranes for immunoblotting. PON1 was detected using the mouse anti-DYKDDDDK Tag monoclonal antibody 9A3 (1:500, Cell Signaling Technology, Danvers, MA, USA) and a goat anti-mouse IgG-HRP (1:1000, Cell Signaling Technology). In addition, α-tubulin was detected by a rabbit anti-α-tubulin polyclonal antibody (1:1000, Cell Signaling Technology) and a goat anti-rabbit IgG-HRP (1:2000, Merck, Darmstadt, Germany). Quantification of protein levels was conducted by densitometry analysis of the immunoreactive bands using the ImageJ software (version 1.54d) [31 ].