STLC‐synchronised mitotic (M) wild‐type (WT), FAM83D
−/− knockout (KO) and FAM83D
GFP/GFP knockin (KI) U2OS cells were subjected to anti‐CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 μm.
STLC‐synchronised mitotic (M) FAM83D
−/− knockout (KO), FAM83D
GFP/GFP knockin (KI) and FAM83D
GFP/GFP(F283A) knockin (FA) U2OS cells were subjected to anti‐CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 μm.
Quantification of CK1α spindle localisation for the cells described in panels (A and B). Cell images denote the measured regions used to calculate the ratios on the box plot. Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***P < 0.0001; ANOVA. Analysis was performed on the indicated number of cells, n = 2.
The cell lines described in (B) were STLC‐synchronised and mitotic cells (M) isolated by shake‐off. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to GFP TRAP immunoprecipitation (IP) and subsequent immunoblotting (IB) with the indicated antibodies.
Schematic illustration of the AdPROM‐mediated degradation of FAM83D. VHL; Von Hippel‐Lindau protein, CUL2; cullin 2, RBX1; RING‐box protein 1, E2; E2 ubiquitin‐conjugating enzyme, aGFP.16; anti‐GFP.16 nanobody.
KI cells were infected with retroviruses encoding either VHL, aGFP.16 or VHL‐aGFP.16. Uninfected cells were used as a control. Cells were lysed and subjected to IB with the indicated antibodies.
The cell lines described in (F) were subjected to anti‐CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 μm.
Data information: All blots are representative of at least three independent experiments.
