Gel from Scientific Research

Wild-type (WT) UGGT1 (lanes 1-6), p.Ala711Val (lanes 7-12), p.Arg1272His (lanes 13-18), and p.Arg1546* (lanes 19-24) were expressed individually or co-transfected with AAT-Z in ALG6/UGGT1/2-/- cells. Once prepared, the cellular lysates were split between whole-cell lysates (WCLs) (20%) to detect total protein, 35% for CRT to isolate monoglucosylated UGGT1 or AAT-Z, and 35% to CRT* to detect any background binding to the recombinant GST-CRT protein. The samples were resolved by 9% SDS-PAGE gel before being probed by western blot. Immunoblots were probed with an anti-FLAG polyclonal antibody to measure the expression and reglucosylation of exogenous UGGT1 (top) and AAT-Z (bottom). The immunoblots were also probed with an anti-UGGT1 polyclonal antibody to ensure bands were specific to overexpressed UGGT1 (middle).

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