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Fig. 2. Expression of EPMH reporters in bloodstream trypanosomes. (A) Control untransformed bloodstream cells (BS221) and cell lines expressing EPMH or EPMH{Delta}gpi were preincubated (1 hour) with (+) or without (-) tunicamycin, and then metabolically radiolabeled with [35S]Met/Cys (1 hour) in the continued absence or presence of inhibitor. Reporters and endogenous VSG were immunoprecipitated from cell and media fractions with anti-EP9 (top, 2x107 cell equivalents/lane) and anti-VSG221 (bottom, 2x105 cell equivalents/lane) antibodies. Mobilities of the precursor (P), and mature (M) forms of EPMH, of EPHM{Delta}gpi ({Delta}), and of VSG (V), are indicated on the left; mobilities of molecular mass markers on the right. (B) EPMH cells were radiolabeled with [35S]Met/Cys for 2 hours and then immunoprecipitated with anti-EP9 (lane 1). Identical anti-EP9 immunoprecipitates were solubilized and reprecipitated with either anti-EP9 (lane 2) or biotinyl-tomato lectin (lane 3). All lanes contain 2x107 cell equivalents/lane. (C) EPMH cells were radiolabeled with [3H]Myristate for 4 hours and labeled polypeptides were immunoprecipitated from cell and media fractions with anti-EP9 (5x107 cell equivalents/lane) or anti-VSG221 (1x106 cell equivalents/lane). All samples were analyzed by SDS-PAGE and phosphorimaging (panels A and B) or fluorography (panel C).





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