Plg-RKT behaves as a regulated integral membrane protein. (A) Membrane fractions or cytoplasmic fractions from either undifferentiated or M-CSF–treated Hoxa9-ER4 cells (30 μg/lane) were electrophoresed on 12% sodium dodecyl sulfate polyacrylamide gels under reducing conditions and Western blotted with either anti–Plg-RKT mAb, anti–α-enolase mAb as a loading control, or isotype control IgG. (B) M-CSF–treated Hoxa9-ER4 cell membranes were solubilized in 3% Triton X-114. After heating at 37°C and separation of the phases by centrifugation, an aliquot of both phases was electrophoresed and Western blotted with anti–Plg-RKT mAb 35B10. In controls for the method, when the cell lysates were spiked with BSA and subjected to phase partitioning, BSA was detected in the aqueous, but not the detergent phase (data not shown).
