Figure 4

Elevated SNX5 does not impact on macropinosome maturation. HEK-FlpIn parental (white bars) or HEK-GFP-SNX5 (black bars) cell monolayers were cultured in the presence of 100 μg/ml Alexa647-conjugated dextran (10,000 MW) for 4 h at 37°C, washed and incubated overnight to label the late endosomes and lysosomes. The cells were then incubated with 100 μg/ml TR-labeled dextran (10,000 MW) for 5 min at 37°C, washed and then incubated at 37°C in serum free media for up to 20 min, as indicated, to internalize the TR-labeled dextran. Monolayers were fixed in 4% PFA and the samples mounted and images captured using a confocal microscope. The proportion of macropinosomes that had fused and acquired content from the late endosomes/lysosomes was scored.