Fig. 7.

Analysis of PM-located BODIPY-SM. CATH.a cells were cooled at 4 °C for 10 min and pulse-labeled with BODIPY-SM (1 μM for B. cereus SMase treatment and 2 μM for the BE protocol) in serum-free culture medium for 30 min at 4 °C. (A) Cells were washed and chased in serum-free culture medium at 37 °C for 60 min to enable BODIPY-SM internalization. Cells were then subjected to B. cereus SMase treatment (150 mU/ml) in serum-free culture medium at 37 °C up to 4 h. Cellular lipid extracts were analyzed by HPLC. (B) After pulse labeling cells were washed and chased at 37 °C. At the indicated times cells were subjected to BE at 4 °C followed by BODIPY-SL analysis in the BE fractions and cell extracts by HPLC. Results shown represent mean values from one representative experiment performed in duplicates.