Fig. 3: TFG is regulated by phosphorylation across the cell cycle.

a Representative micrograph of interphase (left) and mitotic (right) HeLa cells with endogenously tagged TFG. Cells were stained with Hoechst (magenta). Scale bar 10 μm. b Micrographs of recombinant TFG-162-240-mEGFP-FLAG purified from synchronized interphase or mitotic Expi293F cells. Mitosis-derived protein was treated with lambda protein phosphatase (λPP) where indicated. Scale bar 10 μm; inset 500 nm c Schematic depicting predicted phosphorylation sites within TFG (bottom): serine/threonine, orange; tyrosine, cyan, and depicting confidently identified phosphorylation sites found via mass spectrometry in TFG (top) with mitotic phosphorylation site depicted with red/bolded ‘P’. Putative phosphorylation sites were predicted by NetPhos-3.0. d Representative anti-TFG Western blot of PhosTag gel depicting purified recombinant TFG-mEGFP-FLAG derived from synchronized cells in interphase (I) or mitosis (M) as indicated. Arrows indicate species that are less abundant (lowest arrow) or more abundant (upper arrows). Blank lane between each condition was cropped. e Representative micrograph of phosphomimetic TFG-mEGFP-FLAG 24 h post-transfection in HeLa cells. Widefield microscopy, Z-slice, scale bar 10 µm. f Representative widefield deconvolution micrographs of interphase (upper) and mitotic (lower) HeLa cells overexpressing TFG-mEGFP-FLAG (left), TFG-T87A-mEGFP-FLAG (phosphor-dead) (middle), or TFG-T87E-mEGFP-FLAG (phosphomimetic) (right). Cells were stained with Hoechst (magenta). Scale bar 10 µm. 90% of mitotic cells expressing wild-type TFG (n = 24) or TFG-T87E (n = 10) were able to fully dissolve foci, in contrast to 65% in T87A-expressing cells (n = 26). At least three independent experiments were performed to obtain the replicates for each condition. Source data are provided as a Source Data file.