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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: Traffic. 2009 Jul 21;10(10):1502–1517. doi: 10.1111/j.1600-0854.2009.00965.x

Figure 6. β-COP and β-COP/Cog3 knockdowns along with overexpression of Arf1-Q71L cause delays in the SubAB-mediated GRP78 cleavage and block the traffic of SubAB.

Figure 6

GalNAcT2-GFP HeLa cells were mock-transfected or transfected with siRNA to β-COP or β-COP/Cog3. 72 hours after transfection, the cells were solubilized in phosphate-buffered saline with 2% SDS, and the lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies (A). (B) Graph of SubAB-induced GRP78 cleavage for control, β-COP KD or β-COP/Cog3 double-KD cells. (C) Vero cells were transfected with the CFP-Arf1-Q71L plasmid and 24 hours later treated with 1 µg/ml SubAB-HF555 for 20 min. Toxin was additionally internalized for 60 min, after that cells were fixed in 4% paraformaldehyde, permeabilized with Triton X-100, treated with anti-giantin, and developed using HiLyte-647-conjugated anti-rabbit antibodies. Samples were viewed by dual-laser confocal microscopy (63x oil objective). Bar, 10 µm. The asterisk indicates a control cell not transfected with CFP-Arf1-Q71L.