Fig. 7. RNAi of rab4a and rabaptin-5α. (A) HeLa cells were transfected with pKoen (mock), pKoen-rab4a or pKoen-rabaptin-5α for 3 days. Western blots were probed with antibodies against rab4a, rabaptin-5α and tubulin (loading control). In neither case was expression of the other two components of the complex affected. (B) Cells were incubated with 15 µg/ml Alexa-Tf at 16°C for 30 min, chased for different periods of time at 37°C, and processed for fluorescence microscopy. Cells with knocked-down rab4a or rabaptin-5α contain GFP in the nucleus. RNAi of rab4a accelerates exit of Tf from the cells, while reduced rabaptin-5α expression caused a strong inhibition of Tf internalization.