Solubilization of ART2.2-GPI but not ART2.2-Tm by TX-100 is temperature sensitive. DC27.10 cells stably transfected with ART2.2-GPI or ART2.2-Tm were pooled (1 × 106 of each cell type) and lysed in 1% TX-100 for 10 minutes at 4°C (lane 1). Insoluble material was pelleted by centrifugation and solubilized again in 1% TX-100 for 20 minutes at 37°C (lane 2). Insoluble material was pelleted by centrifugation. Pellets were solubilized by ultrasonication in 2% SDS (lane 3). A separate aliquot of pooled ART2.2-GPI– and ART2.2-Tm–transfected DC27.10 cells (1 × 106 of each) was incubated for 60 minutes with PI-PLC. PI-PLC–treated cells were pelleted by centrifugation, and the supernatant was collected (lane 4). The cell pellet was solubilized directly by ultrasonication in SDS (lane 5). Proteins were size fractionated by SDS-PAGE and stained with Coomassie brilliant blue (A). Histone bands in lane 3 are indicated (hi). An identical gel was subjected to immunoblot analysis (B). Immunodetection was performed sequentially by incubation of the blot with peroxidase (PO)–conjugated cholera toxin (GM1), PO-conjugated anti-FLAG mAb M2 (ART2-Tm and ART2-GPI), and rabbit antiserum T20 followed by PO-conjugated anti–rabbit IgG (Gβ). Results are representative of 3 independent experiments.
