Fig. 2. TbG63 is an integral membrane protein with the N-terminus exposed to the cytoplasm. (A) Microsomes from cells stably expressing YFP-TbG63 were washed in HEPES or carbonate buffer in the presence or absence of 1 M KCl. The supernatant (S) and pellet (P) fractions obtained after high-speed centrifugation were analyzed by SDS-PAGE followed by immuno-blotting for YFP and the ER luminal marker, BiP. (B) The same microsomes in (A) were also treated with proteinase K (PK) in the presence or absence of Triton X-100 (TX) and blots probed for YFP and BiP. For both (A) and (B) a representative blot is shown above and quantitation below presented as the mean ± s.d. (n=3). (C) Microsomes from YTat cells were treated as described in A and B, and blots probed for endogenous TbG63 and BiP. (D) Cells transiently transfected with YFP-TbG63 lacking the C-terminus, which includes the predicted transmembrane domain, were fixed and DNA stained using DAPI.