FIGURE 4:

GPI anchoring of E6 reporters. Following TfR silencing, HA:E6- or HA:E6ΔG-expressing cells were lysed at 10⁸ cells/ml in TEN buffer containing 1% NP40. Lysates were incubated at 37°C for 5 min to allow complete hydrolysis of all GPI anchors by the activity of endogenous GPI-specific phospholipase C. Lysates were then immunoprecipitated (IP) as indicated with anti-TfR (αTfR, 10⁷ cell equivalents/precipitate) or anti-VSG (αVSG, 5 × 10⁵ cell equivalents/precipitate) antibodies covalently cross-linked to protein A sepharose. One set (top) of matched anti-TfR precipitates was immunoblotted (IB) with anti-CRD (αCRD), and the other with anti-HA (αHA). Likewise, one set (bottom) of matched anti-VSG precipitates was immunoblotted with anti-CRD (αCRD), and the other with anti-VSG. Images from a representative experiment are presented. Mobilities of E6 subunits, VSG, and molecular mass markers (kDa) are indicated.