Fig. 2. Localisation of activated HA-Dvl-2-ER protein in single-copy cell line. All images represent a projection of a series of stacked optical sections. Nuclei were stained with ToPro III (blue). Areas of colocalisation in overlaid images appear yellow. Cultures were treated with 1 µM ß-estradiol for 1 hour, fixed and stained, except as noted for panels G-I. (A-C) Colocalisation of F-actin and HA-Dvl-2-ER. F-actin is in red (A) and HA-Dvl-2-ER in green (B). Arrowheads in inset image indicate patches of HA staining projecting from the juxtamembrane region into cell interior. Arrowheads in the merged image (C) indicate examples of areas of colocalisation at the cell membrane. The complete series of optical sections is shown in supplementary material Fig. S3. (D-F) Colocalisation of -tubulin and HA-Dvl-2-ER protein with -tubulin in green (D) and HA-Dvl-2-ER in red (E). Arrows in merged image (F) indicate examples of co-staining at MTOC. (G-I) Disruption of membrane localisation of HA-Dvl2-ER protein by nocodazole treatment. Cells were treated with 33 µM nocodazole (a concentration which disrupts microtubule networks but not the MTOC) in DMSO for 3 hours and then with 1 µM ß-estradiol plus nocodazole for up to 1 hour. -tubulin is in green (G) and HA-Dvl-2-ER in red (H). Arrows in merged image (I) indicate examples of co-staining at MTOC. Staining of control coverslips with an anti--tubulin antibody confirmed that microtubule networks were disrupted only in nocodazole-treated cultures and were not affected by the fixation protocol (data not shown). Similar disruption of HA-Dvl-2-ER at the membrane was seen in cells treated with nocodazole for periods of between 2 hours and 15 minutes (data not shown). Bars, 10 µm.