*4.6. Preparation of Whole-Cell Lysates*

Forty-eight hours after transfection, whole-cell lysates were prepared in ice-cold lysis buffer A containing 50 mM Tris/HCl (pH 7.4), 1 mM dithiothreitol, 1% (w/v) Triton X-100, and a protease inhibitor cocktail for general use (Nacalai Tesque) as described previously [58]. The protein concentration of the whole cell lysate was quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as a standard according to the manufacturer's protocol. Before glycosidase treatment, the whole cell lysate samples were incubated with PNGase F (New England Biolabs Japan, Tokyo, Japan) (1.25 U/µg of protein) at 37 ◦C for 10 min as described previously [59], and then subjected to immunoblotting.
