Figure 1.

Characterization of clones stably expressing GFP–Dad1 that is functionally incorporated into the OST. (a) Isolated clones express different levels of GFP–Dad1 and respond differently to the inducer. Permanently transfected clones isolated after antibiotic and nonpermissive temperature selection were grown in the absence (−) and presence (+) of the inducer. Equal protein amounts of total cell lysates were analyzed on Western blots using an anti-GFP pAb. (b) In clone M3/18, the expression level of GFP–Dad1 is comparable to that of endogenous Dad1. Equal amounts of protein from total lysates of cells grown at the permissive or nonpermissive temperature were analyzed on Western blots using an anti-Dad1 pAb. Lane 1, BHK-21 cells grown at 34°C; lane 2, tsBN7 cells grown at 34°C; lane 3, M3/18 cells grown at 34°C and lane 4, M3/18 cells grown at 39.5°C. An asterisk marks the position of GFP–Dad1 protein and the position of endogenous Dad1 is marked by a double asterisk. (c) N-glycosylation activity of OST is repaired in M3/18 cells grown at 39.5°C. Three different cell lines transiently expressing SEAP were grown at 34 or 39.5°C and labeled with [³⁵S]-TransLabel. The cell lysates were immunoprecipitated with anti-SEAP pAb. The precipitates were digested with EndoH (+) or left as untreated control (−). The EndoH-resistant form of SEAP is represented by upper most band (arrow 1), the EndoH-sensitive form of SEAP is a band in the middle (arrow 2), and the nonglycosylated form of SEAP is represented by lower band (arrow 3).