mouse monoclonal anti- apc4 antibody [santa cruz (b- 11) sc. # 5148960]  (Santa Cruz Biotechnology)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The Mitotic Checkpoint Complex controls the association of Cdc20 regulatory protein with the ubiquitin ligase APC/C in mitosis.

doi: 10.1073/pnas.2413089121

Figure Lengend Snippet: Fig. 1. MCC blocks the release of Cdc20 from APC/C by Cdk1-catalyzed phosphorylation. (A) MCC inhibits the release of Cdc20 from APC/C by Cdk1 action. The release of myc-Cdc20 to from APC/C beads to supernatants was determined as described under Materials and Methods. Where indicated, 25 nM recombinant MCC (containing flag-Cdc20) was added, and mixtures were incubated at 23 °C for 15 min with shaking at 1,400 rpm to allow the binding of MCC to APC/C. Subsequently, Cdk1-cyclin B (1,000 units) was added as specified, along with 2 mM ATP, 10 mM phosphocreatine, and 0.1 mg/mL creatine phosphokinase. Following further incubation (23 °C, 1,400 rpm) for the time periods indicated, phosphorylation was stopped by addition of Cdk inhibitor p27T187A (5 μM), Samples of supernatants of APC/C beads were treated with lambda phosphatase as described (22), and myc-Cdc20 was detected by immunoblotting with anti-myc tag antibody. Results are expressed as the percentage of total myc-Cdc20 present in the supernatant. sup., supernatant. The number on the right side indicates the position of the molecular weight marker protein. (B) MCC prevents the loss of Cdc20 from APC/C by Cdk1. Experimental conditions were similar to those described in (A), except that amounts of myc-Cdc20 that remained associated with APC/C beads were determined as described in Materials and Methods. All samples were treated with lambda phosphatase prior to SDS-PAGE and immunoblotting for myc-tag. Immunoblots for APC4 served as loading controls. Numbers on the right indicate the position of molecular weight marker proteins. (C) MCC causes the accumulation of partially phosphorylated forms of APC/C-bound Cdc20. Experimental conditions were as in (B), except that following incubation and washing of APC/C-myc-Cdc20 beads, they were not subjected to lambda phosphatase treatment and were resolved by Phos-tag SDS-PAGE, as described (22). Numbers on the right side indicate the position of molecular weight marker proteins (Bio-Rad), the migration of which was retarded by the Phos-tag reagent. (D) MCC does not protect from phosphorylation N-terminal threonine sites of Cdc20 bound to APC/C. The indicated phosphorylation site mutants of myc-Cdc20 were constructed, expressed, and purified as described in Materials and Methods. Lanes 1 to 3, myc-Cdc20 mutants were bound to APC/C beads and were incubated with Cdk1-cyclin B and MCC as described in (A and B), except that incubations were for 30 min and washed beads were not subjected to phosphatase treatment. Lanes 4 to 5, free myc-Cdc20 mutants were incubated with or without Cdk1 phosphorylation mixture. All samples were subjected to Phos-tag gel electrophoresis and were blotted with myc-tag antibody.

Article Snippet: Loading controls for amounts of APC/C in beads were by immunoblotting with a mouse monoclonal anti- APC4 antibody [Santa Cruz (B- 11) sc.

Techniques: Phospho-proteomics, Recombinant, Incubation, Binding Assay, Western Blot, Molecular Weight, Marker, SDS Page, Migration, Construct, Purification, Nucleic Acid Electrophoresis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The Mitotic Checkpoint Complex controls the association of Cdc20 regulatory protein with the ubiquitin ligase APC/C in mitosis.

doi: 10.1073/pnas.2413089121

Figure Lengend Snippet: Fig. 3. MCC blocks ubiquitylation-dependent release of APC/C-bound Cdc20. (A) Ubiquitylation of Cdc20APC/C promotes its release from APC/C. Myc-Cdc20 was bound to immunopurified APC/C and beads were subjected to further washes prior to incubation as described under “Materials and Methods.” Beads were suspended in 20 μL of incubation mixture (Materials and Methods). Where indicated, a ubiquitylation mixture (“Ub”) consisting of 200 nM E1 ubiquitin-activating enzyme (Enzo Life Sciences), 500 nM UbcH10, 100 mM recombinant human ubiquitin (R & D Systems), and 2 mM AMP-PNP [adenylyl-5′-(β, γ)-imidodiphosphate, Sigma] was added (final concentrations). Following incubation at 23 °C with shaking at 1,400 rpm for the times indicated, samples were taken from total incubation mixture, washed APC/C beads, and supernatants of APC/C beads, as described under “Materials and Methods.” “myc-Cdc20-Ubn,” polyubiquitylated derivatives of myc-Cdc20. Numbers on the right side indicate the position of molecular weight marker proteins (kDa), (B) Quantitation of amounts of derivatives of myc-Cdc20 in DUB-treated supernatants of APC/C beads. Supernatants of APC/C beads from an experiment similar to that described in A were subjected to treatment with the deubiquitylating enzyme (“DUB”) USP2 catalytic domain (R & D Systems), as described in Materials and Methods. Results were quantified and were expressed relative to the amount of myc-Cdc20 in control incubation without ubiquitylation (lane 1). (C) MCC inhibits the ubiquitylation of Cdc20APC/C and its release from APC/C. Experimental conditions were as in (A), except that where indicated, MCC (25 nM) was added and samples were incubated at 23 °C for 15 min prior to the addition of ubiquitylation mixture and further incubation for the time periods indicated. Amounts of free myc-Cdc20 in DUB-treated supernatants were quantified and expressed as in (B). (D) MCC prevents the loss of APC/C-associated Cdc20 due to ubiquitylation. Experimental conditions were as in (C), except that APC/C beads were washed following incubation as described in Materials and Methods and amounts of myc-Cdc20 that remained associated with APC/C were estimated by immunoblotting with Myc-tag antibody. Shown are loading controls of anti-APC4 immunoblots of the same samples.

Article Snippet: Loading controls for amounts of APC/C in beads were by immunoblotting with a mouse monoclonal anti- APC4 antibody [Santa Cruz (B- 11) sc.

Techniques: Incubation, Ubiquitin Proteomics, Recombinant, Molecular Weight, Marker, Quantitation Assay, Control, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Hyperphosphorylation of CDH1 in glioblastoma cancer stem cells attenuates APC/C CDH1 activity and pharmacological inhibition of APC/C CDH1/CDC20 compromises viability

doi: 10.1158/1541-7786.MCR-18-1361

Figure Lengend Snippet: (a) The APC/C holoenzyme was immunoprecipitated (IP) using anti-CDC27 from G1 synchronized NSTCs and CSCs from GBM 3832 with resulting levels of the indicated APC/C subunits identified by silver staining. Control lane = anti-CDC27 antibody and beads only. Antibody heavy chain is indicated (IgG). (b) The APC/C holoenzyme was immunoprecipitated using anti-CDC27 from G1 synchronized NSTCs and CSCs from GBM 3832 with resulting immunoprecipitates analyzed by LC-MS/MS. Protein abundance for the indicated APC/C holoenzyme subunits and CDH1 were quantified and resulting fold change graphed (normalized to the NSTCs). (c-e) Whole-cell lysates from G1 synchronized NSTCs and CSCs from GBM 3832 (c), 3691 (d), and 08-387 (e) were probed for APC4 and CDH1. βactin served as a loading control. (f-h) The APC/C holoenzyme was immunoprecipitated from G1 synchronized NSTCs and CSCs from GBM 3832 using anti-CDC27 (f) or anti-APC4 (g) and from 3691 using anti-APC4 (h). Resulting immunoprecipitates were probed for CDC27 or APC4 and CDH1 (top two panels). Control lane = anti-CDC27 or anti-APC4 antibody and beads only. Bound CDH1 relative to CDC27 or APC4 as an indication of active APC/C was quantified and resulting fold change graphed (normalized to NSTCs). n = 3 biological replicates, error bars represent standard deviation; *P < 0.05; **P < 0.01; ***P < 0.001. Bottom two panels represent inputs for the CDC27 or APC4 immunoprecipitation. βactin or α-tubulin served as loading controls. For all immunoblots, the molecular weight (MW) of resulting bands is given in kilodaltons (kD).

Article Snippet: Immunoprecipitation (all used at 1 ug per reaction); mouse anti-CDC27, clone AF3.1 (Sigma-Aldrich), mouse anti-APC4, clone CIV1.1 (Novus Biologicals), or mouse anti-CDH1, clone DH01 (Calbiochem/MilliporeSigma).

Techniques: Immunoprecipitation, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Western Blot, Molecular Weight

Journal: Molecular cancer research : MCR

Article Title: Hyperphosphorylation of CDH1 in glioblastoma cancer stem cells attenuates APC/C CDH1 activity and pharmacological inhibition of APC/C CDH1/CDC20 compromises viability

doi: 10.1158/1541-7786.MCR-18-1361

Figure Lengend Snippet: (a-c) Whole-cell lysates from G1 synchronized NSTCs and CSCs from GBM 3832 (a), 3691 (b), and 08-387 (c) were probed for EMI1. βactin served as a loading control. (d-e) The APC/C holoenzyme was immunoprecipitated (IP) from G1 synchronized NSTCs and CSCs from GBM 3832 (d) or 3691 (e) using anti-APC4. Resulting immunoprecipitates were probed for APC4 and EMI1 (top two panels). Control lane = anti-APC4 antibody and beads only. The line in (d) indicates that two non-relevant lanes were removed from the image between the control lane and the 3832 immunoprecipitation lanes. Bound EMI1 relative to APC4 was quantified and resulting fold change graphed (normalized to NSTCs). n = 3 biological replicates, error bars represent standard deviation; ns = no significance. Bottom two panels represent input for EMI1 immunoprecipitation. α-tubulin served as a loading control. For all immunoblots, the molecular weight (MW) of resulting bands is given in kilodaltons (kD).

Article Snippet: Immunoprecipitation (all used at 1 ug per reaction); mouse anti-CDC27, clone AF3.1 (Sigma-Aldrich), mouse anti-APC4, clone CIV1.1 (Novus Biologicals), or mouse anti-CDH1, clone DH01 (Calbiochem/MilliporeSigma).

Techniques: Immunoprecipitation, Standard Deviation, Western Blot, Molecular Weight