Fig. 2. BiP is controlled at a translational level. (A) Left, northern blot analysis of poly A⁺ RNA isolated from Bil11 cells activated for various time periods, as indicated. The probe detecting both mouse (m-BiP) and human BiP (h-BiP) corresponds to a 1.1 kb fragment of the mouse BiP coding sequence (which has more than 92% identity to the human sequence). Actin mRNA was probed with the complete actin coding sequence. Right, quantification was performed by phosphoimaging and normalized for actin signals. Relative amounts of endogenous human BiP mRNA (grey bars) and heterologous mouse BiP mRNA (black bars) are presented. (B) Left, determination of the rate of BiP synthesis in Bilu33 cells expressing only endogenous human BiP (h-BiP) and activated Bil11 cells co-expressing human and mouse BiP (t-BiP). Cells were cultured in the absence of tetracycline, and lysates were prepared at time points indicated after ³⁵S-methionine addition. BiP was immunoprecipitated by monoclonal anti-BiP antibody. To directly compare BiP synthesis rates in the different cells, the respective amounts of cell lysates were normalized according to the amount of TCA-precipitated radioactivity determined in the first samples. Right, gels were exposed for the same time period and relative signal intensities of BiP in Bilu33 (grey bars) and Bil11 cells (black bars) were determined by phosphoimaging. (C) Left, kinetics of BiP degradation in Bilu33 expressing only endogenous human Bip (h-Bip) and activated Bil11 co-expressing human and mouse BiP (t-BiP). Cells were pulse labeled for 1.5 hours with ³⁵S-methionine, and identical volumes of culture were taken to prepare lysates at the time points indicated after initiation of the chase. Total BiP was immunoprecipitated by monoclonal anti-BiP antibody and signals quantified by phosphoimaging. Right, the amount of labeled BiP (grey bars: human BiP in Bilu33; black bars: total BiP in Bil11) is expressed as a percentage of labeled BiP isolated directly after the pulse.

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