Fig. 4. Alix depletion alters the distribution of transferrin-positive recycling endosomes. HeLa cells were treated with control RNA (A-D) or with siRNA against Alix (E-H) as in Fig. 1B. The cells were incubated with Tf-Alexa⁵⁹⁴ at 37°C for 5 minutes followed by a 15-minute chase to label the recycling endosomes (A,E). The cells were fixed before permeabilisation and were stained with anti-EEA1 (B,F) and anti-Rab11 (C,G). D and H are merged images of B,C and F,G, respectively. White colour indicates colocalisation between Tf-Alexa⁵⁹⁴, EEA1 and Rab11. Scale bars: 5 µm.