Fig. 4. UPR induction by thapsigargin and effect of actinomycin D on BiP synthesis in activated Bil11 cells. (A) Determination of ³⁵S-methionine incorporation into TCA-precipitated material from lysates of thapsigargin- (black bars) or mock- (DMSO-)treated cells (gray bars) prepared at the time points indicated after ³⁵S-methionine addition. (B) Upper panel, determination of BiP (t-BiP) synthesis rate was performed as described in Fig. 3C. Lower panel, relative signal intensities of total BiP in thapsigargin- (black bars) or mock- (DMSO-)treated (grey bars) cells. (C) Upper panel, determination of mouse BiP synthesis rate was performed as described in Fig. 3D. Lower panel, relative signal intensities of mouse BiP isolated from thapsigargin- (black bars) or mock-treated (gray bars) cells. (D) Determination of ³⁵S-methionine incorporation into TCA-precipitated material from lysates of activated Bil11 cells treated with thapsigargin during starvation (90 minutes) and pulse, and additionally with actinomycin D 5 minutes prior to and during pulse (black-checkered bars). Mock- (DMSO-)treated cells are also shown (gray bars). (E) Upper panel, determination of BiP (t-BiP) synthesis rate was performed as described in Fig. 3C. Lower panel, relative signal intensities of total BiP in thapsigargin and actinomycin D- (black-checkered bars) or mock (DMSO)-treated (grey bars) cells. (F) Upper panel, determination of mouse BiP synthesis rate was performed as described in Fig. 3D. Lower panel, relative signal intensities of total BiP in thapsigargin and actinomycin D- (black-checkered bars) or mock- (DMSO-)treated (grey bars) cells.

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