American Society of Hematology

Fig. 4.

$Fig. 4. The protease is in a membrane bound subcellular compartment and is in excess to PI-6. (A) U937 cells were fractionated into cytosol and Triton X-100 soluble membrane fractions as described in Materials and Methods. Equal amounts of cytosolic protein, membrane protein, and a 1:1 mixture of both were incubated at 37°C for 10 minutes. Samples were separated by 10% SDS-PAGE under reducing conditions and immunoblotted using polyclonal anti–PI-6 antisera to visualize the 56-kD complex. The 63-kD band present in the first two lanes is nonspecific and was recognized by preimmune sera (not shown). (B) U937 cells were lysed and incubated at 37°C for 10 minutes with or without 2 ng recombinant PI-6 (rPI-6). Samples were separated by 10% SDS-PAGE under reducing conditions and immunoblotted using polyclonal anti–PI-6 antisera. Two nanograms of recombinant PI-6 was included in a separate lane as a positive control.$

The protease is in a membrane bound subcellular compartment and is in excess to PI-6. (A) U937 cells were fractionated into cytosol and Triton X-100 soluble membrane fractions as described in Materials and Methods. Equal amounts of cytosolic protein, membrane protein, and a 1:1 mixture of both were incubated at 37°C for 10 minutes. Samples were separated by 10% SDS-PAGE under reducing conditions and immunoblotted using polyclonal anti–PI-6 antisera to visualize the 56-kD complex. The 63-kD band present in the first two lanes is nonspecific and was recognized by preimmune sera (not shown). (B) U937 cells were lysed and incubated at 37°C for 10 minutes with or without 2 ng recombinant PI-6 (rPI-6). Samples were separated by 10% SDS-PAGE under reducing conditions and immunoblotted using polyclonal anti–PI-6 antisera. Two nanograms of recombinant PI-6 was included in a separate lane as a positive control.

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