Figure 4

BtpB subcellular localisation. HeLa cells were transiently transfected with a plasmid encoding GFP-BtpB. Sixteen hours after transfection, cells were left untreated (A-D), or treated (E, F) with 1 μg/ml nocodazole for 30 min, fixed and stained with mouse anti β-tubulin antibody, followed by detection with anti-mouse Texas Red. (A). Confocal images of BtpB and the microtubule network. From left to right, merged stack images of 8 Z-slices of overlay, and individual GFP-BtpB (green) and tubulin (red) images are shown. Scale bar 10 μm. (B). Images, from left to right, show the individual Z-slices (0,3 μm depth) from the cell presented in A. The arrow head in panel 4 indicates GFP-BtpB accumulation in the nucleus. Boxed areas in panels 4 and 6 are shown enlarged in C and D. (C) Individual image (red filter) showing microtubule network at the MTOC (the lines indicate the area of GFP-BtpB colocalisation, as shown in the overlay image below). (D) Partial association of BtpB punctae and tubulin indicated with arrows. Enlargement (10x). (E) and (F) Confocal slices of HeLa cells after nocodazole treatment (+NC). Arrows indicate individual cells showing complete protection of nocodazole-induced microtubule depolymerization in transfected cells. Boxed areas are shown enlarged. Scale bar 10 μm. (E) Arrow head in enlarged image points at GFP-BtpB accumulation in the Midbody of the intercellular bridge.