Figure 2.

NtWLIM2 Subcellular Localization in BY-2 Cells. (A) Typical distribution of NtWLIM2–GFP in stably transformed BY-2 cell lines. Beside a clear nuclear localization (arrow), NtWLIM2–GFP interacted with filamentous cytoplasmic structures, later identified as the actin cytoskeleton (Supplemental Figure 2). (B) Modification of NtWLIM2–GFP subcellular distribution following Lat B treatment. Transgenic NtWLIM2–GFP cells were treated with 2.5μM Lat B and GFP fluorescence distribution was monitored over 25min. A notable increase of nuclear signal is observed upon Lat B treatment. Arrows point to prominent cables (upper panel), which disappeared after Lat B treatment (lower panel).(C) Quantification of GFP fluorescence in the nucleus of BY-2 cells after 25-min mock or Lat B treatment (white column and black column, respectively). GFP fluorescence intensity in the nucleus after 25min of mock or Lat B treatment was determined in a defined area using ImageJ software and normalized to the GFP fluorescence measured in the same area at the start of the time course. Error bars denote the 95% confidence interval obtained for these GFP fluorescence ratios (n=23 for Lat B-treated cells, n=7 for mock-treated cells, p-value obtained by Student’s t-test=1.27e–12). Image panel: typical GFP fluorescence intensity in the nucleus of BY-2 cells before (upper panels) and after treatment with mock buffer (control, left panels) or Lat B (right panels). Scale bars: 5μm.(D) Immunodetection of endogenous NtWLIM2 (left) by anti-WLIM2 antibody in isolated BY-2 nuclei and nucleoli (see Supplemental Figure 3 for the specificity of this antibody). DAPI counterstain (middle panel) enabled localization of WLIM2 (left panel) relative to the nuclear shape in the merged picture (right panel). Scale bar: 5μm.