Fig. 1. BiP expression is tightly controlled in unstressed cells. (A) Left,
detection of mouse BiP in lysates of Bil11. Bil11 cells were cultured in the
presence of tetracycline, and lysates were prepared from equal amounts of
cells taken at time points indicated after removal of tetracycline.
Equivalents of 105 cells were loaded. The western blot was
developed with anti-GRP78 antiserum, which detects only heterologous mouse BiP
(m-BiP), and with anti-tubulin antibodies. Right, tubulin was used for
normalization of the total cellular proteins loaded. Amounts of mouse BiP
(black bars) were expressed as a percentage of steady state amounts of mouse
BiP present in permanently activated cells. (B) Left, western blot analysis of
total BiP (t-BiP) performed on one tenth of the lysates used in A. The blot
was developed with monoclonal anti-BiP antibody detecting both human and mouse
BiP equally and with anti-tubulin antibodies. Right, tubulin was used for
normalization of the cellular proteins loaded. Amounts of total BiP (grey
bars) were expressed as a percentage of steady state levels of total BiP
present in permanently activated cells. (C) Left, quantification of mouse and
total BiP in permanently activated cells. The amounts of lysate indicated were
prepared from permanently activated cells and mixed with defined amounts of
purified recombinant BiP-GST fusion protein (BiP/GST), as indicated. Mouse or
total BiP was detected by using anti-GRP78 antiserum (upper panel) or
monoclonal anti-BiP antibody (lower panel), respectively. Note that the
BiP-GST fusion protein contains the epitopes for both anti-GRP78 antiserum and
monoclonal anti-BiP antibody. Right, calculation of the ratio of mouse BiP
according to the amount of total BiP after removal of tetracycline. Amounts of
mouse BiP and total BiP giving signals in the linear range were assigned to a
corresponding amount of BiP-GST fusion protein, allowing the determination of
the ratio of mouse BiP to total BiP in permanently activated cells. The
gradual replacement of human BiP (gray bars) by mouse BiP (black bars) after
removal of tetracycline was monitored.