Fig. 3.

MICAL-L1 inhibits cell surface delivery of TNFα and E-cadherin but not that of GPI. HeLa cells expressing Scr and two different shRNAs targeting MICAL-L1 were transfected with TNFα-SBP-mCherry (TNFα), cadherin-mCherry (cadherin)t and GPI-mCherry (GPI) using the RUSH system. Cells were incubated with biotin at 37°C for 45 min for TNFα and GPI and 90 min for cadherin. Cells were washed with cold PBS at 0°C, Incubated with anti-mCherry at 0°C for 30 min, followed by another incubation with AlexaFluor647 donkey anti-rabbit for 30 min at 0°C to detect cargoes at the cell surface. Cells were then fixed and analyzed by confocal microscopy. The ratio of cell surface/total fluorescence was quantified, at least 40 cells for each condition were analyzed.