FIGURE 8:

GPI-anchor status of HA:E6*. Following TfR silencing (24 h), HA:E6-expressing cells were incubated with MG132 (2 h) to block proteasomal degradation. Cells were then detergent-lysed under conditions that either subject all GPI anchors to hydrolysis by endogenous GPI-PLC (PLC+) or preserved GPI integrity (PLC-). (A) Lysates (10⁷ cell equivalents) were immunoprecipitated with mouse anti-HA cross-linked to Protein G Sepharose and sequentially immunoblotted with rabbit anti-CRD (top panel) followed by mAb anti-HA (bottom panel), generating matched signals from the same membrane. (B) Lysates (5 × 10⁵ cell equivalents) were immunoprecipitated with rabbit anti-VSG cross-linked to Protein A Sepharose and then immunoblotted with rabbit anti-CRD (top panel) or mAb anti-VSG (bottom panel), generating matched signals from the same membrane. Blots from a single representative experiment are presented in A and B. (C) Total (T), cytosolic (C), and membrane (M) fractions were prepared as in Figure 7A. HA:E6 polypeptides were immunoprecipitated (10⁷ cell equivalents) with anti-HA and then blotted with anti-CRD (top) followed by anti-HA (bottom), generating matched signals from the same membrane. Mobilities of intact HA:E6 (E6), protected deglycosylated HA:E6 (star), and VSG (V) are indicated on the left in A–C; mobilities of molecular mass markers on the right.