Figure 7.

MICAL-L1 regulates the recycling of Tf receptor and β1 integrins back to the plasma membrane. (A–D) HeLa cells on coverslips were mock-treated (A and B) or treated with MICAL-L1-siRNA (C and D). Seventy-two hours later the cells were serum-starved for 30 min and pulsed with Tf-568 for 10 min and fixed (A and C), or first “chased” in complete medium for 25 min at 37°C before fixation (B and D). (E) Quantitative analysis of the percentage of internalized Tf remaining after 15 min of pulse and 40 min of chase was done. Approximately 80 cells from three independent experiments were counted for each set of treatments. Error bars, SE. (F–I) HeLa cells on coverslips were mock-treated (F and G) or treated with MICAL-L1-siRNA (H and I) for 72 h. Cells were then starved and pulsed with anti-β1 integrin antibody for 1 h and fixed (F and H) or pulsed for 1 h and chased in complete media for 3 additional hours at 37°C before fixation (G and I). (J) Quantitative analysis of the percentage of β1 integrins remaining after 3 h of chase was done as described for Tf. Significance (p < 0.05) was determined by the Student's t test for independent samples. Bar, 10 μm.