Western Blot Analysis of LuloHya Protein

Western blot was carried out as described elsewhere [9 (link)]. Briefly, the content of 5 Lu. longipalpis SG and 100 ng of LuloHya were separated by NuPAGE protein gels. Proteins were transferred to a nitrocellulose membrane (iBlot, Invitrogene) that was blocked overnight at 4°C with blocking buffer: 50 mM Tris, 150 mM NaCl containing 5% (w/v) powdered nonfat blotting-grade milk (Bio-Rad) and 0.05% of Tween-20 (Sigma). Mouse anti-LuloHya antibodies were diluted in blocking buffer (1:5,000) and incubated for 90 min. Goat anti-mouse conjugated to alkaline phosphatase (Sigma) diluted in blocking buffer (1:10,000) was used as a secondary antibody. Immunogenic protein bands were visualized by Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega, Madison, WI) and reaction was stopped with distilled water.