Fig. 2. (A) Western blot of T7-VSG 117 (wild-type VSG 117 expressed in the same context as the mutants) compared with mutants S505G, S505A, S505T, K510R and K510L. The cells were induced with 100 ng/ml doxycycline for 24 hours, and the equivalent of 2x10⁴ cells were loaded per lane. (B,C) Western blots of cell line T7-VSG 117 after endogenous GPIPLC activation. Aliquots representing 2x10⁴ cells were loaded per lane. Cells coexpressing VSGs 117 and 221 were osmotically lysed in ice-cold water and centrifuged. The cell ghosts (P1) were resuspended and incubated in 10 mM sodium phosphate buffer, pH 8.0, at 37°C for 15 minutes and centrifuged to generate pellet (P2) and supernatant (S) fractions. Lanes C, P1, P2 and S contain, respectively, whole cells, pellet after lysis and pellet and supernatant after GPIPLC activation. (B) VSG 117 release in the absence or presence of 5 mM pCMPS during PIPLC cleavage, probed with antibodies to VSG 117. (C) Analysis of the same samples, in the absence of pCMPS, with anti-VSG 221 antibodies. (D) Analysis with anti-BiP antibodies. (E) The same protocol was applied to a GPIPLC null-mutant cell line expressing VSG 221 (Leal et al., 2001) and the blot was probed with VSG 221 antibodies.