Fig. 9. Effect of Sar1-depleted cytosol on the presence of COPII proteins in PCTVs. (A) Native cytosol (Cy), Sar1 immunodepleted cytosol (Sar1 dep Cy), native ER, and ER washed with 2 M urea (Urea-treated ER) (30 µg protein each) were probed with anti-Sar1 antibodies. (B) PCTVs (30 µg protein), formed under different conditions as indicated, were used for immunoblots as in Fig. 2. PCTVs formed in native cytosol (Cy) or from Sar1 immunodepleted cytosol (Sar1 dep Cy) or in the presence of H89 (H89) were probed with anti-Sar1 antibodies. PCTVs (30 µg protein) formed in native cytosol (Cy) or Sar1 immunodepleted (Sar1 dep Cy) cytosol were probed with antibodies to Sar1, Sec24, Sec31, syntaxin 5, p58 and rBet1. (C) The effect of protease treatment on PCTV cargo (apoB-48) generated in Sar1-depleted cytosol. PCTVs generated in Sar1 depleted cytosol were incubated with proteinase K (0.5 mg/ml) as in 5B. 30 µg protein of protease treated or untreated PCTVs were separated on 7.5% SDS-PAGE and immunoblotted with rabbit polyclonal anti-apoB-48 antibodies. For details, see Materials and Methods.