Figure 5

Immunoprecipitation experiments indicate that the intracellular, immature OSTα and OSTβ are physically associated. (A) Lysates from COS7 cells transfected with OSTα-FLAG and OSTβ-Myc for 48 hrs were subjected to immunoprecipitation using mouse anti-FLAG agarose beads (left two panels) or rabbit anti-Myc and proteinA/G beads (right two panels). Following PAGE and transfer to PVDF membrane the blots were probed with rabbit antibodies to OSTα (top left panel) and OSTβ (bottom left panel) or mouse antibodies to FLAG (top right panel) and His (bottom right panel), respectively. As noted in the Methods, OSTβ-Myc also contains a His tag. Anti-FLAG agarose efficiently removes all OSTα-FLAG from the lysate (Lys), however, it only co-precipitates a portion of OSTβ(left two panels). Although anti-Myc was rather inefficient in removal of OSTβ-Myc from the lysate, it was capable of co-precipitating the immature forms of OSTα(right two panels). The mature, glycosylated OSTα (arrowhead) was never detected in the precipitate. (B) COS7 cells transfected with OSTα alone, OSTβ alone, or both subunits were subjected to metabolic labeling and immunoprecipitation. Twenty-four hours after transfection cells were pulsed for 15 min with ³⁵S-Trans label. Cells were either lysed immediately (0 chase) or chased for 2 hrs (120 chase). Immunoprecipitation was carried out using mouse anti-FLAG agarose beads (left panel) or rabbit anti-Myc and proteinA/G beads (right panel). The left panel shows that the higher molecular weight band (glycosylated form of OSTα) is only present in cells transfected with both subunits, and only after the 2 hr chase (arrowhead). A time course indicated that it was detectable after 30 min of chase (data not shown). The ~20 kD OSTβ subunit is co-precipitated from these cells (+). In contrast, immunoprecipitation of OSTβ with anti-Myc antibody resulted in co-precipitation of only the immature form of OSTα (right panel). The asterick (*) indicates the position where the mature form would have appeared.