Endos binding and inhibition of PP2A-Tws depends mainly on its Gwl phosphorylation site. (A) D-Mel cells transfected with the indicated proteins were submitted to GFP-Tws immunoprecipitation (IP), and products were analyzed by WB for GFP and Endos. (B) The indicated variants of GST-Endos (or GST alone) were tested for their ability to pull down Tws-Flag from a cell extract (detected with anti-Flag). (C) Summary of Endos variants tested in A and B for their interaction with Tws. (D) The ability of the indicated GST-Endos variants to inhibit PP2A-Tws phosphatase activity toward a phosphopeptide was quantified. (E and F) C-terminal fusion of the SV40 NLS to Endos does not prevent its ability to bind Tws (E, GST pulldown as in B) or to inhibit PP2A-Tws (F, phosphatase assay as in D). Bars: mean ± SD, n = 3. **, P = 0.0022; ****, P ≤ 0.0001. WCE, whole-cell extract.