Spatiotemporal regulation of Tws localization (complements to Figs. 1 and 2). (A) Left: D-Mel cells expressing Tws-Flag were treated with 50 nM LMB or 0.1% ethanol (EtOH; mock control) for 4 h before fixation, immunostaining, and DNA staining with DAPI. Scale bar: 5 µm. Right: Quantifications of the nuclear/cytoplasmic ratio of signal intensities (mean ± SD). (B) WB from third instar larvae in which expression of the indicated Tws variants was driven by Ubi-Gal4. (C) All GFP-fused Tws variants can associate with PP2A-C (microtubule star [Mts]) and PP2A-29B. 0–2-h-old embryos expressing the indicated proteins were submitted to GFP-affinity purifications. Purification products were analyzed by WB. (D) Images of larval brains (ventral nerve cord) from third instar larvae expressing Tws variants. DNA was stained with Hoechst 33342. Note the higher nuclear/cytoplasmic ratio of NLSm^SV40-Tws-GFP (arrows) compared with the other variants. Scale bar: 10 µm. (E) Fusion of NLS^SV40 to Tws does not prevent its ability to bind Endos. A GST pulldown was done as in Fig 3 B. (F) Fusion of NLS^SV40 to Tws does not prevent its ability to be inhibited by Endos^S68D. A phosphatase assay was done as in Fig 3 D. Top: quantification of the phosphatase activity (mean ± SD, n = 3). Bottom: Visualization of the immunoprecipitated (IP) PP2A complexes used in the reactions. Error bar: SD. ****, P ≤ 0.0001. Purif., purification; WCE, whole-cell extract.