Fig. 6. Soluble proteins are secreted from GBF1-depleted cells. (A-C) NRK cells were transfected with reagent alone (`mock' lanes) or with siRNA against GBF1 (`siRNA' lanes) for 72 hours. Untransfected cells were treated with 5 µg/ml BFA for 30 min (`+BFA' lanes). All cells were pulsed for 30 minutes with 35S-Met/Cys and then chased for 4 hours. At that point, media were collected and cells were lysed. Aliquots of media (B) and lysates (A) were analysed by SDS-PAGE and fluorography. Secretion from BFA-treated cells is inhibited, but proteins are efficiently secreted from control and GBF1-depleted cells. Aliquots of lysates were western blotted to confirm GBF1 depletion (C). (D,E) NRK cells were transfected with scrambled siRNA (`scramble' lanes) or with siRNA against GBF1 (`siRNA' lanes) for 72 hours. Untransfected cells were treated with 5 µg/ml BFA for 30 min (`+BFA' lanes). All cells were pulsed for 30 minutes with 35S-Met/Cys and chased for the indicated times. At each time point, media were collected and cells were lysed. Aliquots of media and lysates were precipitated with acid to obtain total protein-incorporated radioactivity. The amount of secreted protein at each time is represented as a percentage of the radioactivity in the medium and the lysate. Secretion from BFA-treated cells is inhibited, but proteins are efficiently secreted from control and GBF1-depleted cells; n=3 (error bars represent the s.d.). Aliquots of lysates were western blotted to confirm the depletion of GBF1 (E). (F-J) HeLa cells were transfected and treated as in A-C. Secretion from BFA-treated cells is inhibited, but proteins are efficiently secreted from control and GBF1-depleted cells. Aliquots of lysates were western blotted to confirm the depletion of GBF1 (H). Media (from G) were immunoprecipitated with antibodies against fibronectin and laminin, and the precipitates were analysed by SDS-PAGE and fluorography (I,J). Fibronectin and laminin are efficiently secreted from GBF1-depleted cells. (K-N) HeLa cells were transfected and labelled as in D,E. At each time point, media were collected and cells were lysed. Media and lysates were immunoprecipitated with antibodies against fibronectin, and the precipitates analysed by SDS-PAGE and fluorography (K-M). Fibronectin is not secreted from BFA-treated cells but is secreted efficiently from GBF1-depleted cells. Aliquots of lysates were western blotted to confirm depletion of GBF1 (N). (O) HeLa cells were transfected and labelled as in D-E. At each time point, media were collected and cells were lysed. Aliquots of media and lysates were immunoprecipitated with antibodies against fibronectin. The precipitates were analysed by SDS-PAGE and fluorography and quantitated by densitometry. The amount of secreted fibronectin at each time is represented as a percentage of the fibronectin in the medium and the lysate. Proteins are efficiently secreted from control and GBF1-depleted cells; n=2 (error bars represent the s.d.).