FAM83D regulation during the cell cycle: A: Nocodazole-synchronised mitotic FAM83DGFP/GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation (IP), followed by incubation ± λ-phosphatase.Figure 4A: Nocodazole-synchronised mitotic FAM83DGFP/GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation (IP), followed by incubation ± λ-phosphatase. Asynchronous (AS) cells were used as a control. Whole cell extracts (input) and IP samples were subjected to SDS-PAGE and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium (B) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 (C) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME (D). AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. E: Wild-type U2OS cells were either left asynchronous (AS), or arrested in mitosis with STLC and collected by shake-off (M). AS and M cells were incubated in media containing combinations of RO-3306 and MG132 as indicated, prior to lysis. MG132 was applied for 1.5 h, whereas RO-3306 was applied for the last 1 h of incubation prior to lysis. Samples were lysed and extracts were subjected to SDS-PAGE, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. F: STLC-synchronised mitotic wild-type U2OS cells were subjected to qRT-PCR analysis using primers for FAM83D, HMMR, CSNK1A1, and CCNB1. AS cells were used as a control. Error bars, SEM; *, P<0.01; Student's T-test. n=3 G: Schematic representing the predicted effects of HMMR knockout on FAM83D-CK1α delivery to the mitotic spindle. In the absence of HMMR, no FAM83D and by extension no CK1α, can localise to the spindle. H: STLC-synchronised mitotic wild-type (WT) and HMMR knockout (KO) mouse embryonic fibroblasts (MEFs) were lysed and subjected to IB with the indicated antibodies. AS cells were included as a control. Data information: All blots are representative of at least 3 independent experiments. The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-HMMR antibody (I) DNA is stained with DAPI. Scale bars, 20 µm. The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-CK1α antibody (J). DNA is stained with DAPI. Scale bars, 20 µm.