TbERP4 mediates GPI-dependent trafficking in PCF cells. TbERP4 mediates GPI-dependent trafficking in PCF cells. (A) The tetracycline-inducible PCF TbERP4 RNAi cell line was cultured with or without tetracycline as indicated. Cell density was measured by hemacytometer every 24 h, and cells were adjusted back to the starting density (5 × 105 cells/ml) every 48 h. Mean ± SEM values from triplicate cultures (for each treatment) are presented. (B and C) PCF TbERP4 RNAi cells containing the BiPN or BIPN:GPI reporters were cultured for 48 h with and without tetracycline as indicated to induce dsRNA. Cells were pulse (10 min)-chase (3 h) radiolabeled, and BiP polypeptides were specifically immunoprecipitated from whole-cell lysates at the indicated chase times and analyzed by SDS-PAGE/phosphorimaging (1 × 107 cell equivalents per lane). (Top) Quantification of BiPN or BiPN:GPI normalized to T0 (mean ± SEM, n = 3). (Bottom) Representative phosphorimages of cell-associated BiP polypeptides. B, native BiP; N, BiPN; M, mature BiPN:GPI; I, immature precursor BiPN:GPI. Emilia K. Kruzel et al. mSphere 2017; doi:10.1128/mSphere.00282-17