Figure 4

Effect of digitonin extraction on distribution of CLIC1 in Panc1 cells. Panc1 cells were fixed with PLP without (A, C) or with (B, D) prior extraction with digitonin. Cells were then stained with AP1089 (A, B) or with both AP1089 and 9F5 (C, D) and imaged using confocal microscopy. In A and B, images are shown from the very base of the cell (left panel) plus images at focal planes 2 (center) and 4 (right) μm higher. Collection of images in B required higher sensitivity than A. Parallel cultures stained with control antisera and imaged under identical conditions were blank (not shown). In panels C and D, cells were double stained with AP1089 with Alexafluor565-conjugated goat anti-rabbit IgG (red, left panel) and 9F5 with Alexafluor488-conjugated goat anti-mouse IgG (green, center panel) without (C) or following (D) digitonin extraction. A merged image for each pair is shown on the right. Scale bar in A and B represent 25 μm, scale bars in C and D represent 20 μm.