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Fig. 5. Cytosol immunodepleted of PKC ${zeta}$ does not support PCTV budding but does support protein-vesicle budding. (A) PCTVs were budded from ER using native cytosol and ER treated with IgG (NC), cytosol immunodepleted of PKC ${zeta}$ (–PKC ${zeta}$ ), or cytosol immunodepleted of PKC ${zeta}$ to which either 2.5 µg recombinant PKC ${zeta}$ (+rPKC ${zeta}$ ) or 2.5 µg PKC ${alpha}$ (+rPKC ${alpha}$ ) was added. For NC, ER treated with 10 mM HEPES, pH 7.2 was used. When PKC ${zeta}$ -depleted cytosol was used, the accompanying ER was treated with 2 M urea. Data are the mean ± s.e.m., n=4. P values indicate differences between the means of –PKC ${zeta}$ and NC or +rPKC ${zeta}$ , or the difference between +rPKC ${alpha}$ and NC or +rPKC ${zeta}$ . (B) PCTV and protein vesicles were budded from [¹⁴C]TAG and [³H]protein loaded ER using cytosol treated with IgG (NC) or cytosol immunodepleted of PKC ${zeta}$ (–PKC ${zeta}$ ). After incubation (Materials and Methods), PCTV and protein vesicles were separated on a continuous sucrose gradient. The gradient was resolved into 20 fractions of 0.5 ml each. The first three fractions were considered to be PCTV and fractions 8 to 10 were considered to be protein vesicles. The TAG was extracted from the PCTV-containing fractions and the proteins collected from the protein-vesicle fractions after TCA precipitation. PCTV-budding activity is shown on the left and protein-vesicle budding activity on the right.

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