Fig. 3. Kinetics of upregulation of BiP expression during UPR. (A) Activated Bil11 cells expressing endogenous human and additional mouse BiP were treated with tunicamycin for 8 hours, and RNA was prepared at the different time points indicated. A northern blot of poly A⁺ RNA was hybridized with the BiP-specfic probe (also used in Fig. 2A), detecting mouse BiP/luciferase mRNA (mBiP/luc) and endogenous human BiP mRNA. Quantification was performed by phosphoimaging and normalized for actin signals. Black bars represent mouse BiP/luciferase mRNA, and grey bars represent human BiP mRNA. (B) Determination of global protein synthesis. DMSO or tunicamycin (in DMSO) was added to activated Bil11 cells for 1.5 hours prior to addition of ³⁵S-methionine. Lysates were prepared from equivalent amounts of cells harvested at the time points indicated after ³⁵S-methionine addition. For determination of the rate of total protein synthesis, incorporation of ³⁵S-methionine into TCA-precipitated material from lysates of tunicamycin- (black bars) or mock-treated (gray bars) cells was measured. (C) Upper panel, for determination of the rate of BiP synthesis, monoclonal anti-BiP antibody was used for immunoprecipitation of total (human and mouse) BiP (t-BiP). Lower panel, relative signal intensities of total BiP isolated from lysates of tunicamycin- (black bars) or mocktreated (gray bars) cells. (D) Upper panel, for determination of the rate of mouse BiP (m-BiP) synthesis, anti-BiP antiserum was used for immunoprecipitation of mouse BiP. Lower panel, relative signal intensities of mouse BiP isolated from lysates of tunicamycin- (black bars) or mock-treated (gray bars) cells.