Fig. 4. TbG63 localizes to the Golgi complex. (A) Biochemical fractionation. (i) Cells (1x10¹⁰) stably expressing the Golgi marker YFP-GntB were homogenized (H), centrifuged at medium speed to generate a pellet (P1) and a supernatant (S1) that was then centrifuged at higher speed to generate a final microsomal pellet (P2) and supernatant (S2). (ii) The microsomal pellet (P2) was further fractionated by isopycnic sucrose gradient centrifugation. For both i and ii equal fractions were analyzed by blotting for the indicated markers and the results quantitated and presented as the mean ± s.d. (n=4). Blots were probed for: Golgi (YFP-GntB), COPI coats (-COP), flagella (PAR), ER (BiP), tubulin (Tub) and TbG63. (B) Fluorescence microscopy. Cells stably expressing YFP-GntB (top row) or YFP-GRIP (bottom row) were fixed and labeled for TbG63 and stained with DAPI to visualize the nucleus (N) and kinetoplast (K). Scale bar: 1 µm.