Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Cloned PC12 cell lines expressing FLAG-Rab5:Q79L and FLAG-Rab5:S34N. Shown are two cell lines expressing FLAG-Rab5:S34N and two lines expressing FLAG-Rab5:Q79L as indicated. The parental PC12 cell line serves as a negative control (lane PC12). The protein expression was determined in the presence or absence of Dox (1 μg/ml) and cell lysates were subjected to immunoblot analysis with anti-FLAG and anti-Rab5 antibodies as indicated. The actin level in each sample is presented as the loading control (anti-actin). Molecular mass standards are indicated on the left (in kDa).

  Initially the expression level was generally high, but decreased with each passage and eventually stabilized at a level comparable or lower than the endogenous level after five passages, which made it difficult to identify with the Rab5 antibody. Thus we tagged both Rab5 mutants with the FLAG epitope to facilitate their detection by immunoblot analysis with the FLAG antibody (Supplemental figure 1, top panel). We also did immunoblot analysis of the same cell lysates with the Rab5 antibody, which showed that the FLAG-tagged Rab5 proteins were expressed at lower level and migrated slightly slower than endogenous Rab5 on SDS-PAGE (Supplemental figure 1, middle panel). Occasionally there were some cell clones whose expression remained high after five passages (e.g., the #2 clone of Q79L in Fig. 3A), but even these clones eventually reduced the Rab5 mutant expression to a level similar to the other clones with additional passages. The reduction in expression was likely an adjustment and adaptation by the PC12 cells rather than loss of cell population expressing the recombinant protein, because this phenomenon was observed in both Q79L-expressing cells that showed faster cell growth and S34N-expressing cells that showed slower cell growth (Figure. 4). In addition, the whole cell population showed similar Rab5 mutant expression level as evidenced by immunofluorescence microscopy with the FLAG antibody (data not shown).