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Table I.

Cytosolic protein is required for ATP-independent transport of ceramide



Transport
Addition
+ATP
−ATP

 
(%)
 
 
Untreated cytosol 100 100 
No cytosol (B88) 
No cytosol (B88 containing BSA) 
Treated cytosol   
+ Trypsin, 30 min, 4°C, + trypsin inhibitor 16 
+ Trypsin + trypsin inhibitor, 30 min, 4°C 87 117 
15 min, 95°C 
Untreated cytosol + 1% Triton X-100 100 ND 
No cytosol (B88) + 1% Triton X-100
 
99
 
ND
 


Transport
Addition
+ATP
−ATP

 
(%)
 
 
Untreated cytosol 100 100 
No cytosol (B88) 
No cytosol (B88 containing BSA) 
Treated cytosol   
+ Trypsin, 30 min, 4°C, + trypsin inhibitor 16 
+ Trypsin + trypsin inhibitor, 30 min, 4°C 87 117 
15 min, 95°C 
Untreated cytosol + 1% Triton X-100 100 ND 
No cytosol (B88) + 1% Triton X-100
 
99
 
ND
 

Cytosolic protein is required for ATP-independent transport of ceramide. In vitro ceramide transport assay was performed with untreated or treated cytosol in the presence or absence of ATP as described in the legend to Fig. 5 B. For trypsin treatment, cytosol was treated with trypsin (1.25 mg/ml) for 30 min at 4°C and quenched with soybean trypsin inhibitor (5 mg/ml). In control treatments, soybean trypsin inhibitor was added before addition of trypsin. For heat treatment, cytosol was incubated for 15 min at 95°C and centrifuged for 1 min at 12,000 g, and the resulting supernatant was used for the assay. If present, Triton X-100 (1%) was added to reaction mixtures for 20 min at 4°C before the reaction. Lipid analysis was performed and ceramide transport (%) was determined as described in the legend to Fig. 5 C. ND, not determined.

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