Journal: bioRxiv
Article Title: High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells
doi: 10.1101/2024.06.20.599917
Figure Lengend Snippet: (A) Heatmap showing canonical protein abundance profile for experimentally validated APC/C recognition motifs in both Time Course and Mitotic Exit datasets. Protein changes are coloured according to their abundance (log 2 palbociclib arrest normalised values). Degradation motifs are indicated with different colours. Proteins not detected are shown with a dashed box. (B) The proportion of CCD proteins with experimentally validated APC/C recognition motifs and established degradation mechanisms (purple) versus those without a known mechanism (light grey). Proteins found to contain unstable peptides in a global peptide stability dataset are also shown in dark grey. (C) Heatmap showing protein abundance changes for CCD proteins predicted to contain a degradation motif with high confidence and selected for validations. Protein changes are coloured according to their abundance (log 2 palbociclib arrest normalised values). (D) Representative western blot analysis (n=3) of predicted mitotic APC/C substrates degradation in Hela Kyoto cells during the override of a mitotic checkpoint arrest by the CDK1 inhibitor RO-3306 in the presence and absence of ANAPC4. SGO2, GTSE1, MELK, FAM38D and known APC/C substrates (CCNB1) are stabilised by proteasome inhibition with MG132.
Article Snippet: After 1 h blocking with 5% milk in TBST at room temperature (RT) for 1 h, membranes were incubated overnight at 4°C with the following primary antibodies: CCND1 (Santa Cruz Biotechnology, sc-2004), CCND3 (Santa Cruz Biotechnology, sc-6283), CCNE2 (Santa Cruz Biotechnology, sc-248), CCNA (Santa Cruz Biotechnology, sc-271682), CCNA (Santa Cruz Biotechnology, sc-596), CCNB1 (Santa Cruz Biotechnology, sc-245), GAPDH (Santa Cruz Biotechnology, sc-51907), CDK1 pT161 (Cell Signalling Technology, 9114), CDK1 pY15 (Cell Signalling Technology, 9111), Alpha Tubulin (Abcam, ab52866), PLK1(Millipore, 05-844), CDK2 (Santa Cruz Biotechnology, sc-163), CDK1 (Abcam, ab32094), CDC20 (Santa Cruz Biotechnology, sc-13162), UBR7 (Thermo Fisher Scientific, A304-130A), UBE2S (Cell Signalling Technology, 11878), CDKN1B (Dako, M7203), CDKN1B pS10 (Santa Cruz Biotechnology, sc-12939), NCL (Abcam, ab22758), NCL pT76 (Abcam, ab168363), CCNB1 (Santa Cruz Biotechnology, sc-245), SGO2 (Thermo Fisher Scientific, A301-261A), GTSE1 (Abnova, H00051512-B01P), MELK (Thermo Fisher Scientific, A303-136A), FAM83D (Custom antibody from Erich Nigg), ANAPC4 (Thermo Fisher Scientific, A301-175A), Phospho-CDK substrate motif [(K/H)pSP] (Cell Signalling Technology, 9477), JUNB (Proteintech, 10486-1-AP), CHAF1B (Thermo Fisher Scientific, A301-085A), Histone H3 pS10 (Cell Signalling Technology, 3377), Actin (Sigma-Aldrich, A5441).
Techniques: Western Blot, Inhibition