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Fig. 7. Anti-VAMP7 antibodies inhibit the transfer of TAG but only modestly reduce the transfer of newly synthesized proteins from the ER to the cis Golgi. ³H-TAG-loaded rat intestinal ER (300 µg protein) were incubated with 10 µl pre-immune IgG (A), ER treated with 10 µl of anti-VAMP7 antibody (B), cis Golgi treated with 10 µl anti-VAMP7 antibodies (C), ER treated with anti-syntaxin8 antibodies (D) or ER treated with anti-Sec22b antibodies (E). Unbound antibody was removed by washing and the ER was incubated with 1 mg native rat cytosol, 500 µg rat cis Golgi, an ATP-generating system and buffer B for 30 minutes at 35°C. The cis Golgi was isolated on a sucrose step gradient (see Materials and Methods) and obtained by aspiration. TAG was extracted and the dpm determined. The data are the mean ± s.e.m., n=4. There was a significant difference in ³H-TAG transfer (P<0.01) between VAMP7 antibody-treated ER and the pre-immune control. (B) ER (500 µg protein) loaded with ¹⁴C-TAG and ³H-protein was treated with pre-immune IgG (10 µl) (A and C), or anti-VAMP7 antibody (10 µl) (B and D), excess antibody was removed by washing, and incubated with native rat intestinal cytosol (1 mg protein), an ATP-generating system and cis Golgi (500 µg protein). The Golgi fraction was isolated on a sucrose gradient and the amount of TAG (left y axis) or protein (right y axis) dpm determined (Materials and Methods). The percentage reduction in transport upon VAMP7 antibody treatment is shown above the bars. A compared with B, P<0.001; C compared with D, P>0.05. The data are the mean ± s.e.m. (n=4).

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