mouse monoclonal anti cdc27 apc3  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: CCT5 maintains mitotic fidelity and promotes early colorectal tumorigenesis

doi: 10.1016/j.isci.2026.115223

Figure Lengend Snippet: CCT5 maintains the structural integrity of the MCC-APC/C complex and facilitates APC/C activation (A) Co-immunoprecipitation (CoIP) analysis of CDC20-associated proteins follows release from double thymidine block. CDC20 was immunoprecipitated, and the association of mitotic checkpoint complex (MCC) components, including BUBR1, CDC27, BUB3, and MAD2L1, was examined by immunoblotting. Representative immunoblots from n = 3 independent biological experiments are shown (n represents independent experiments). Reduced association of MCC components with CDC20 was observed in CCT5-depleted cells. (B) Co-immunoprecipitation (CoIP) analysis of APC/C-associated proteins follows release from double thymidine block. APC3 was immunoprecipitated, and the association of MCC components, including BUBR1, CDC27, CDC20, BUB3, and MAD2L1, was examined by immunoblotting. Representative immunoblots from n = 3 independent biological experiments are shown (n represents independent experiments). Reduced association of MCC components with the APC/C complex was observed in CCT5-depleted cells, while input protein levels remained comparable. (C and D) Cycloheximide (CHX) chase assay shows reduced stability of CDC20 in CCT5-deficient HCT116 cells. Representative immunoblots are shown in (C). Quantitative densitometric analysis of CDC20 protein levels normalized to GAPDH and expressed relative to the 0-h time point is shown in (D). Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA (group × time) followed by Šídák’s multiple comparisons test. ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (E) RT-qPCR analysis of CDC20 mRNA expression follows CCT5 knockdown in HCT116 cells. No significant change in CDC20 mRNA levels was observed. Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test. ns, not significant. (F) Cell viability assay assesses whether CDC20 overexpression rescues the proliferation defect induced by CCT5 knockdown in HCT116 cells. Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA (group × time) followed by Šídák’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CDC20 overexpression did not restore proliferation in CCT5-depleted cells. (G and H) Flow cytometry analysis of cell cycle distribution in HCT116 cells follows CCT5 knockdown and CDC20 overexpression. Representative histograms are shown in (G). Quantification of the G2/M fraction is shown in (H). Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance for the G2/M fraction was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. ∗∗ p < 0.01; ns, not significant. CDC20 overexpression did not restore the G2/M phase distribution in CCT5-depleted cells. (I and J) Ubiquitination assay shows increased CDC20 ubiquitination following CCT5 knockdown in HCT116 cells under asynchronous conditions (nocodazole −, MG132 +). CDC20 was immunoprecipitated, and ubiquitinated CDC20 was detected by immunoblotting with anti-ubiquitin antibodies (I). Quantitative densitometric analysis of ubiquitinated CDC20 normalized to immunoprecipitated CDC20 is shown in (J). Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was assessed using an unpaired t test. ∗∗ p < 0.01. (K and L) Ubiquitination assay shows reduced CDC20 ubiquitination following CCT5 knockdown in HCT116 cells under nocodazole-induced mitotic arrest conditions (NOC +, MG132 +). CDC20 was immunoprecipitated and ubiquitinated. CDC20 was detected by immunoblotting with anti-ubiquitin antibodies (K). Quantitative densitometric analysis of ubiquitinated CDC20 normalized to immunoprecipitated CDC20 is shown in (L). Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was assessed using an unpaired t test. ∗ p < 0.05.

Article Snippet: Mouse monoclonal anti-CDC27 (APC3) , Santa Cruz Biotechnology , Cat# sc-9972, RRID: AB_627228.

Techniques: Activation Assay, Immunoprecipitation, Blocking Assay, Western Blot, Quantitative RT-PCR, Expressing, Knockdown, Two Tailed Test, Viability Assay, Over Expression, Flow Cytometry, Ubiquitin Proteomics

Journal: Nature communications

Article Title: APC/C-mediated ubiquitylation of extranucleosomal histone complexes lacking canonical degrons.

doi: 10.1038/s41467-025-57384-7

Figure Lengend Snippet: Fig. 1 | APC3 binds nucleosome acidic patch through HMGN-like nucleosome binding motif. a Electrostatic surface of nucleosome (PDB 3AFA), left, and zoomed view of acidic patch showing residues mutated in acidic patch interaction screen, right. b Profile plots of APC/C subunits and HMGN1 for triplicate wild-type and mutant nucleosome affinity proteomics experiments (data from41). Individual quantitated peptides shown in grey and weighted averages in red or blue. ΔAP: H2A E61A, E64S, N68A, D72S, N89A, D90A, E91S. Additional profile plots shown in Supplementary Fig. 1. c Pulldowns from HEK293T nuclear lysates using recon- stituted, wild-type or mutant biotinylated nucleosomes followed by western blot

Article Snippet: A western blot was later performed with the same membrane using a mouse anti-APC3 antibody (1:10,000, Santa Cruz, SC-9972) and 680CWgoat anti-mouse IgG secondary antibody (1:10,000, LI-COR) allowing simultaneous detection of the label transfer reagent and APC3.

Techniques: Binding Assay, Mutagenesis, Western Blot

Journal: Nature communications

Article Title: TRIP13 and APC15 drive mitotic exit by turnover of interphase- and unattached kinetochore-produced MCC.

doi: 10.1038/s41467-018-06774-1

Figure Lengend Snippet: Fig. 4 TRIP13 and APC15 are required for turnover of MCC produced in interphase. a Upper: Schematic of experiment to measure the dependence of the observed mitotic exit delay on kinetochore-catalyzed MCC production. Lower: mitotic timing in reversine-treated TRIP13AID /APC15KO cells in the presence or absence of IAA. (n = 50 for each experimental condition). b Upper: Schematic of experiment to measure the dependence of the observed mitotic exit delay on Mad2 and BubR1. Lower: mitotic timing in Mad2 or BubR1 RNAi treated TRIP13AID/APC15KO cells in the presence or absence of IAA. (n = 50 for each experimental condition). c Upper: Schematic of experiment to measure the dependence of the observed mitotic exit delay on Tpr. Lower: mitotic timing in Tpr RNAi treated TRIP13AID /APC15KO cells in the presence or absence of reversine and IAA. (n = 50 for each experimental condition). d Upper: Schematic of experiment to measure APC/CMCC formation in TRIP13AID/APC15KO. APC3 was immunoprecipitated from cells in G1 with Palbociclib and in late G2 with RO-3306. Lower: APC/C interacting BubR1 and Cdc20 levels were measured by quantitative immunoblotting. Lines in a–c represent the mean ± s.e.m.

Article Snippet: Rabbit antiBubR1 (homemade, 1:10000), rabbit anti-TRIP13 (Bethyl, Cat# A303–605, 1:3000), rabbit anti-Mad2 (Bethyl, Cat# A300-301A, 1:3000), mouse anti-p31comet (Mad2L1BP) (Santa Cruz, Cat# SC-134381, 1:200), mouse anti-Cdc20 (Santa Cruz, Cat# SC-136024, 1:200), mouse anti-APC3 (Cdc27) (Santa Cruz, Cat# SC-9972, 1:200), mouse anti-GAPDH (6C5) (Abcam, AB8245, 1:10000), and mouse anti-αtubulin (DM1α) (Abcam, AB7291, 1:10000) were used for immunoblotting.

Techniques: Produced, Immunoprecipitation, Western Blot