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Fig. 4. Secretory transport of tsO45-G-YFP is not significantly inhibited following Sec13 depletion. Cells depleted of Sec13 or lamin A/C were infected with an adenovirus expressing tsO45-G-YFP followed by incubation at the restrictive temperature of 39.5°C for 18 hours. (A) Transport of tsO45-G-YFP through the secretory pathway in cells transfected with either lamin-A/C or Sec13 siRNA. Upper panels: YFP (total tsO45-G) fluorescence 60 minutes after the shift to 32°C shows indistinguishable Golgi localization and plasma-membrane delivery in either experiment; note the enhanced juxtanuclear clustering of the Golgi in Sec13-suppressed cells. Lower panels: shows only plasma-membrane-localized tsO45-G-YFP 90 minutes after the shift to 32°C. Scale bars: 20 µm. (B) Quantification of arrival of tsO45-G-YFP at the plasma membrane (monitored by immunolabelling paraformaldehyde-fixed but non-permeabilized cells with an antibody against an extracellular epitope); error bars show standard deviation (n=100 cells from three independent experiments). (C,D) Arrival of protein at the Golgi following release of cargo from the ER at 32°C for the times indicated was determined by monitoring acquisition of EndoH resistance and immunoblotting for YFP. Samples were also immunoblotted to confirm the efficacy of Sec13 depletion and -tubulin was included as a loading control. Molecular-mass markers are shown in kDa.

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