rabbit anti apc8  (Bethyl)

Journal: Cell Reports

Article Title: Deletion of APC7 or APC16 Allows Proliferation of Human Cells without the Spindle Assembly Checkpoint

doi: 10.1016/j.celrep.2018.10.104

Figure Lengend Snippet: APC16 Is Required for Assembly of APC7 into APC/C (A) Experimental setup for MS-based analysis of APC/C composition. APC8-mCherry-expressing wild-type cells were labeled with medium SILAC, and ΔAPC7 or ΔAPC16 cells were labeled with heavy SILAC. APC/C was purified using mCherry affinity beads, and as a control, a mock pull-down was performed from light SILAC-labeled cells. (B) SILAC ratios for APC/C subunits enriched from APC8-mCherry wild-type and APC8-mCherry ΔAPC7 cells. APC/C was purified with mCherry pull-downs from wild-type (medium SILAC) and ΔAPC7 cells (heavy SILAC) and analyzed by MS. Mock pull-down from wild-type HCT116 cells served as a control (light SILAC). The table shows combined SILAC ratios for APC/C subunits detected in three technical replicates (n.d., not determined). (C) Analysis of APC/C composition in ΔAPC7 cells. APC/C was purified via APC8-mCherry pull-downs from APC8-mCherry and APC8-mCherry ΔAPC7 cells (from two independent ΔAPC7 clonal cell lines) and subsequently analyzed by immunoblotting using the indicated antibodies. Wild-type cells were used as a control for unspecific binding to the affinity beads. GAPDH levels were analyzed to verify equal amounts of input for the different cell lines. (D) SILAC ratios for APC/C subunits enriched from APC8-mCherry and APC8-mCherry ΔAPC16 cells. The analysis was performed as described in (B). (E) Immunoblot analysis of APC/C composition in ΔAPC16 cells with the indicated antibodies. APC/C was purified via APC8-mCherry pull-downs from APC8-mCherry cells and from APC8-mCherry ΔAPC16 cells. The indicated samples were transiently transfected with APC16-EGFP 32 hr prior to APC/C pull-down. Wild-type cells were used as a control for unspecific binding to the affinity beads. See also Figure S1 .

Article Snippet: Anti-APC8 (D5O2D) Rabbit mAb , Cell Signaling Technology , Cat# 15100.

Techniques: Expressing, Labeling, Multiplex sample analysis, Purification, Control, Western Blot, Binding Assay, Transfection

Journal: Cell Reports

Article Title: Deletion of APC7 or APC16 Allows Proliferation of Human Cells without the Spindle Assembly Checkpoint

doi: 10.1016/j.celrep.2018.10.104

Figure Lengend Snippet: Analysis of In Vitro Ubiquitylation Activities of APC/C Lacking APC16 and/or APC7 (A) Ubiquitylation activity of purified APC/C variants toward securin. APC/C was purified from APC8-mCherry (WT), ΔAPC7 APC8-mCherry (ΔAPC7), or ΔAPC16 APC8-mCherry (ΔAPC16) cells using an antibody against APC3 and incubated for different times with recombinant securin supplemented with UBE2C and UBE2S as indicated. Ubiquitylation of securin was analyzed by an α-securin immunoblot. A representative result from two experiments is shown. WT, wild-type; DN, dominant-negative. (B) Immunoblot analysis of the purified APC/C used for Figure 2 A. Input, supernatant, and immunoprecipitated fractions (immunoprecipitation [IP]: APC3) from the indicated cell lines were analyzed with the indicated antibodies. (C) Ubiquitylation activity of purified APC/C variants toward cyclin B1 as described in (A). Ubiquitylation of cyclin B1 was analyzed by an α-cyclin B1 immunoblot. A representative result from two experiments is shown. (D) Immunoblot analysis of the purification of APC/C used for Figure 2 C, analyzed as described in (B). See also Figure S1 .

Article Snippet: Anti-APC8 (D5O2D) Rabbit mAb , Cell Signaling Technology , Cat# 15100.

Techniques: In Vitro, Activity Assay, Purification, Incubation, Recombinant, Western Blot, Dominant Negative Mutation, Immunoprecipitation

Journal: Cell Reports

Article Title: Deletion of APC7 or APC16 Allows Proliferation of Human Cells without the Spindle Assembly Checkpoint

doi: 10.1016/j.celrep.2018.10.104

Figure Lengend Snippet: Analysis of Mitotic APC/C Function in ΔAPC7 and ΔAPC16 Cells (A) Time between nuclear cyclin B1 influx and anaphase onset for the indicated cell lines. Wild-type, ΔAPC7, and ΔAPC16 cell lines expressing mVenus-tagged histone H2B and mCerulean3-tagged cyclin B1 were imaged every 5 min. The data show the time from nuclear cyclin B1 influx to anaphase onset for individual cells. Cells with identical timing are depicted as a box, and the size of each box is scaled according to the percentage they contribute to the whole population of the respective cell line. The mean timing and SD are stated on the top. The median timing is drawn as a black line. Results of three independent experiments are shown, and the number (n) of analyzed cells is stated for each cell line. A two-tailed t test was performed to calculate significance (p < 0.01 = significant; n.s., non-significant). (B) Cyclin B1 degradation rates around anaphase onset in the indicated cell lines (data from the experiment shown in A). The plot shows cyclin B1 degradation rates for individual cells from the indicated genetic backgrounds. Cyclin B1 degradation rates in individual cells were calculated by the slope of a linear fit for the measured decrease in cyclin B1-mCerulean3 intensities around anaphase onset. The number of analyzed cells is given as n. The median cyclin B1 degradation rate is drawn as a black line. A two-tailed t test was performed to calculate significance (p < 0.01 = significant). The table on the right shows the mean cyclin B1 degradation rate and its SD in the indicated cell lines. (C) SILAC ratios for APC/C and MCC subunits detected in MS analysis of APC8-mCherry pull-down with or without SAC activation. APC/C was purified via APC8-mCherry pull-downs from untreated cells (medium SILAC) and from cells treated with 200 nM nocodazole (noc) for 18 hr (heavy SILAC). Depicted are the combined SILAC ratios from three technical replicates. A control pull-down from light SILAC was used as a reference for unspecific binding to the affinity beads. (D) SILAC ratios for APC/C and MCC subunits detected in MS analysis of APC8-mCherry pull-down from APC8-mCherry ΔAPC7 cells, analyzed as described in (C). (E) The indicated cell lines were treated with control small interfering RNA (siRNA) or MAD2-targeting siRNA for 96 hr, stained with Hoechst, and imaged. Representative images from one of the three independent experiments are shown. The scale bar indicates 10 μm. (F) Quantification of normal nuclei from the experiments described in (E). From each experiment, at least 500 cells were analyzed per condition, and the cumulative percentage of normal nuclei from all three experiments is shown. Error bars depict SD from 3 experiments. See also and .

Article Snippet: Anti-APC8 (D5O2D) Rabbit mAb , Cell Signaling Technology , Cat# 15100.

Techniques: Expressing, Two Tailed Test, Multiplex sample analysis, Activation Assay, Purification, Control, Binding Assay, Small Interfering RNA, Staining

Journal: Cell Reports

Article Title: Deletion of APC7 or APC16 Allows Proliferation of Human Cells without the Spindle Assembly Checkpoint

doi: 10.1016/j.celrep.2018.10.104

Figure Lengend Snippet: Loss of APC7 or APC16 Provides Synthetic Viability to MAD2 Deletion (A) Strategy for assessing the synthetic viability of MAD2 deletion with the ΔAPC7 genotype. Equal numbers of wild-type, APC8-mCherry, and APC8-mCherry ΔAPC7 cells were mixed and seeded together, resulting in a mixed cell population with one-third of cells lacking APC7. The genomic MAD2 locus was then targeted by CRISPR/Cas9 using a drug-selectable donor plasmid designed to disrupt the MAD2 gene. After selection, each clonal cell line was analyzed by immunoblotting to assess loss of MAD2 and to determine its parent-of-origin cell line. (B) Analysis of APC7 and MAD2 expression in the six clonal ΔMAD2 cell lines retrieved from the setup outlined in (A). GAPDH levels served as a loading control. Note that all six retrieved clonal cell lines derive from APC8-mCherry ΔAPC7 cells. (C) Scheme of the CRISPR/Cas9-based MAD2-GFP synthetic viability assay. Cells expressing endogenously GFP-tagged MAD2 (MAD2-GFP) were transfected with a drug-selectable deletion cassette for the MAD2 gene along with a drug selection-less plasmid encoding a guide RNA targeting either APC7 or APC16. After drug selection, cell colonies were microscopically inspected for loss of MAD2-GFP fluorescence. Cell colonies lacking green fluorescence were then analyzed by immunoblotting for loss of MAD2 expression. The status of APC7 and APC16 in MAD2-deficient cells was assessed by immunoblotting or sequencing of the genomic locus, respectively. (D) Immunoblot analysis of the four clones retrieved from the MAD2-GFP synthetic viability assay performed in combination with an APC7 targeting guide RNA. Note that all retrieved clonal cell lines lost expression of APC7. (E) Analysis of SAC functionality in ΔAPC7ΔMAD2 and ΔMAD2-GFPΔAPC16 cells. Cellular DNA from wild-type, ΔAPC7ΔMAD2, and ΔMAD2-GFPΔAPC16 cells, with or without 18 hr of 200 nM nocodazole, was stained with propidium iodide and analyzed by flow cytometry. MAD2 depletion by RNAi was performed in wild-type HCT116 cells for comparison. The tables at the top show the percentage of cells with the respective (2N, 2N-4N, 4N, and >4N) DNA content. The bottom panel shows the corresponding flow cytometry profiles from the indicated cell lines. See also Figure S4 .

Article Snippet: Anti-APC8 (D5O2D) Rabbit mAb , Cell Signaling Technology , Cat# 15100.

Techniques: CRISPR, Plasmid Preparation, Selection, Western Blot, Expressing, Control, Viability Assay, Transfection, Fluorescence, Sequencing, Clone Assay, Staining, Flow Cytometry, Comparison

Journal: Cell Reports

Article Title: Deletion of APC7 or APC16 Allows Proliferation of Human Cells without the Spindle Assembly Checkpoint

doi: 10.1016/j.celrep.2018.10.104

Figure Lengend Snippet:

Article Snippet: Anti-APC8 (D5O2D) Rabbit mAb , Cell Signaling Technology , Cat# 15100.

Techniques: Transduction, Virus, Recombinant, Transfection, Negative Control, Software