Table 1.
Quantification of native Rabaptin 5/Rabex 5 complex
| A. Total amounts in cells | |||
|---|---|---|---|
| Rabaptin 5 | Rabex 5 | Rab5 | |
| Cytosol | 32 ± 13 pmol/mg | 5 ± 3 pmol/mg | 6 ± 3 pmol/mg |
| Early endosomes | 9 ± 2 pmol/mg | 6 ± 2 pmol/mg | 115 ± 61 pmol/mg |
| B. Stoichiometry of complex in cytosol | Rabaptin 5∶Rabex 5
|
|||
|---|---|---|---|---|
| Immunoprecipitation | Rabaptin 5 | Rabex 5 | Ratio | Average |
| α Rabaptin 5 | 1549 U | 258 U | 6.0∶1 | 5.9 ± 0.2∶1 |
| α Rabex 5 | 439 U | 212 U | 2.1∶1 | 2.1 ± 0.1∶1 |
A. Total Rabaptin-5, Rabex-5, and Rab5 in cells. Different quantities of sHeLa cytosol (10–700 μg) and enriched early endosomes (10 and 20 μg) were loaded on a 12% SDS-PAGE gel. In addition, 5–100 ng of recombinant Rabaptin-5, Rabex-5, or Rab5 isolated from insect cells were loaded as standards. The proteins were then transferred to nitrocellulose and probed by Western with Rabaptin-5, Rabex-5, or Rab5 specific antibodies. Several exposures on x-ray films were obtained and scanned, and the native proteins were quantified using a titration curve obtained with the recombinant standards. Only the exposures falling into the linear part of the titration curves were considered. The data are expressed per milligram of total sHeLa protein as measured with a Biorad protein assay kit. B. Stoichiometry of the native Rabaptin-5/Rabex-5 complex in cytosol. Subconfluent HeLa cells were metabolically labeled with 35S met/cys. Cytosol was prepared from the cells and split in four equal samples. Following a preclearing step, the complexed and free Rabaptin-5 or Rabex-5 molecules were immunoprecipitated with affinity-purified anti-Rabaptin-5α or Rabex-5 antibodies, respectively. The samples were analyzed by SDS-PAGE and were quantified on a Fujifilm FLA2000 Phosphor Fluorescent Imager. The arbitrary counts (units) were corrected for the methionine and cysteine contents of the proteins. “Ratio” refers to the signal obtained for Rabaptin-5 divided by the one obtained for Rabex-5 for the experiment shown in the table. To the right, “average” indicates the results from two independent experiments (± standard error).