Fig. 5. (A) PCTVs and protein vesicles concentrate SNARE proteins as analyzed by immunoblotting of different subcellular fractions for syntaxin 5 and p58. Proteins (30 µg from each fraction) were separated on 12% SDS-PAGE and immunoblotted as described. (B) The effect of protease treatment on a PCTV cargo protein. PCTVs were incubated with proteinase K (0.5 mg/ml) in the presence or absence of 1% Triton X-100 for 30 minutes at 4°C. 30 µg of protein from treated or untreated PCTVs were used for immunoblotting for apoB-48. (C) Immunoblots of subcellular fractions for Sar1, Sec13 and Sec31. Proteins (30 µg for each fraction except PCTVs, 10 µg) from the different subcellular fractions were separated on 12% SDS-PAGE (Sar1 and Sec13) and 10% SDS-PAGE (Sec31), followed by immunoblotting as in Fig. 2. (D) Immunoblot showing depletion of Rab1 from cytosol (Rab1 dep cy) by using anti-Rab1 antibodies and depletion of Rab1 from the ER (Rab-GDI ER) by treatment with Rab-GDI. For details, see Materials and Methods.