phospho-cdk1 substrate antibody  (Millipore)

Journal: eLife

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.7554/eLife.59999

Figure Lengend Snippet: ( A, B ) HeLa cells expressing GFP-CHMP7 δHelix6 were imaged live ( A ) and the intensity of GFP-CHMP7 δHelix6 puncta was quantified during M-phase ( B ). Quantification of 59 cells from nine experiments. Fluorescence traces normalised to metaphase onset (open arrowhead); nuclear envelope breakdown indicated by closed arrowhead. ( C ) Lysates of HeLa cells stably expressing GFP-CHMP7 and subject to the indicated synchronisations were immunoprecipitated using GFP-trap resin and resolved using normal or PhosTag SDS-PAGE. Inputs and captured fractions were examined by western blotting with antisera raised against GFP, or CDK1 substrate consensus sequences ([K/H]-[pS]-[P] or [pS]-[P]-[X]-[R/K]). Western blots representative of 6 PhosTag immunoprecipitations. ( D ) Lysates of HeLa cells stably expressing GFP-CHMP7 or GFP-CHMP7 S3A, S441A and subject to the indicated synchronisations were immunoprecipitated using GFP-trap resin and resolved using normal or PhosTag SDS-PAGE. Inputs and captured fractions were examined by western blotting with antisera raised against GFP, Histone H3 pSer10 or GAPDH (N = 3). Figure 3—source data 1. GFP-CHMP7 delta-Helix6 cluster disassembly during M-phase.

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Expressing, Fluorescence, Stable Transfection, Immunoprecipitation, SDS Page, Western Blot

Journal: eLife

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.7554/eLife.59999

Figure Lengend Snippet: ( A ) 293 T cells expressing the indicated GFP-CHMP7 constructs were cultured asynchronously or synchronised in prometaphase with STLC. GFP-tagged proteins were immunoprecipitated and examined by western blotting with antisera raised against GFP and the CDK1 substrate consensus sequence [K/H]-[pS]-[P]. N = 3. ( B ) HeLa cells stably expressing GFP-CHMP7 393-CT or GFP-CHMP7 393-CT S441A were cultured asynchronously or synchronised prometaphase with STLC, then were lysed, resolved by normal or PhosTag SDS-PAGE and examined by western blotting with antisera raised against GFP, pSer10 Histone H3 or GAPDH (N = 3). ( C, D ) 293 T cells expressing the indicated GFP-CHMP7 NT proteins were cultured asynchronously or synchronised in prometaphase with STLC, then were lysed, resolved by normal or PhosTag SDS-PAGE and examined by western blotting with antisera raised against GFP or GAPDH (N = 3). In C, Ser/Thr containing mutants selected from a previously generated panel of alanine-scanning GFP-CHMP7 NT mutations were assessed for a mobility shift in mitosis. Mobility shift was compromised by M1, M27, and M28, although M27 and M28 were poorly expressed. M1 encodes the mutations W2A, S3A, P4A, E5A; M27 encodes the mutations R106A, E107A, S108A, D109A; M28 encodes the mutations F110A, M111A, A112G, S113A. Individual mutations of S3A, S108A, and S113A from M1, M27, and M28 revealed the site of phosphorylation to be Ser 3 ( D ).

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Expressing, Construct, Cell Culture, Immunoprecipitation, Western Blot, Sequencing, Stable Transfection, SDS Page, Generated, Mobility Shift, Phospho-proteomics

Journal: eLife

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.7554/eLife.59999

Figure Lengend Snippet: ( A ) Recombinant CHMP7 was incubated with recombinant CDK1 and CCNB1 in the presence or absence of ATP and sedimented by ultracentrifugation. CHMP7 was recovered from pellet and supernatant fractions, respectively, resolved by PhosTag SDS-PAGE and analysed by western blotting using antisera raised against CHMP7. ( B ) Data from A presented as mean ± S.D., N = 4, *** p = 0.0007 by paired two-tailed T-test. ( C ) Recombinant CHMP7, CHMP7 S3D/S441D, or CHMP7 S3E/S441E were sedimented by ultracentrifugation, recovered from pellet and supernatant fractions, and analysed by western blotting with antisera raised against CHMP7. ( D ) Data from C presented as mean ± S.D., N = 8, *** p = 0.0004. ( E ) Recombinant CHMP7 was incubated with recombinant CDK1 and CCNB1 in the presence or absence of ATP. Recombinant HA-LEM2 CT was added and CHMP7 was captured on magnetic anti-CHMP7 Dynabeads. Captured and input fractions were resolved by SDS-PAGE and examined by western blotting with anti-CHMP7 or anti-HA antisera. ( F ) Data from G are presented as mean ± S.D., N = 7, p = 0.0156 by paired two-tailed T-test. ( G ) Recombinant HA-LEM2 CT was incubated alone, with CHMP7 or CHMP7 S3D/S441D. CHMP7 was captured on magnetic anti-CHMP7 Dynabeads. Captured and input fractions were resolved by SDS-PAGE and examined by western blotting with anti-CHMP7 or anti-HA antisera. Please note non-specific bands detected by CHMP7 antibody (*). ( H ) Data from G are presented as mean ± S.D., N = 6, p < 0.0001 by paired two-tailed T-test. ( I ) Recombinant LEM2 CT , CHMP7, or CHMP7 S3D/S441D were examined by negative stain electron microscopy, either alone or in the combinations indicated. When incubated alone, no examples of polymerisation were observed. Images representative of N = three independent experiments. Scale bar is 25 nm. Figure 4—source data 1. Influence of CDK1-phosphorylation on the CHMP7/LEM2 interaction.

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Recombinant, Incubation, SDS Page, Western Blot, Two Tailed Test, Staining, Electron Microscopy, Phospho-proteomics

Journal: eLife

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.7554/eLife.59999

Figure Lengend Snippet: ( A, B ) CAL-51 cells homozygously edited to express mNG-CHMP7 ( A ) or HeLa cells stably expressing LEM2-mCh ( B ) were synchronised to prometaphase with STLC and then released through addition of RO-3306. Cells were imaged live through synchronised M-exit. ( C ) Quantification of assembly onset (assembly duration presented in ) post RO3306 release. mNG-CHMP7 +/+ (onset 16.3 ± 0.88 mins; duration 3.1 ± 0.44 mins; N = 9, n = 104) or LEM2-mCh (onset 16.2 ± 2.22 mins; duration 4.1 ± 0.44 mins; N = 3, n = 80). Additional quantification of the above parameters from HeLa cells stably expressing GFP-CHMP7 (onset 16.9 ± 1.21 min; duration 3.3 ± 0.13 min; N = 3, n = 97) and subject to STLC arrest and RO3306 release (from ) and CAL-51 cells homozygously edited to express mNG-CHMP7 (onset 16.1 ± 2.02 min; duration 4.2 ± 2.19 min; N = 7, n = 39) and subject to nocodazole arrest and RO3306 release (from ) are also presented in C. A one-way ANOVA with Tukey’s multiple comparisons revealed no significant differences between the datasets. ( D ). ( E ) HeLa cells stably expressing GFP-CHMP7 NT ( D ) or GFP-CHMP7 ( E ) were either untreated or arrested in mitosis through STLC inhibition and forced out of mitosis using RO3306 for the indicated times. Lysates in D, reporting Ser3 dephosphorylation were resolved by PhosTag or normal SDS-PAGE and examined with antisera raised against GFP or GAPDH respectively. Lysates from E, reporting Ser441 dephosphorylation were immunoprecipitated using GFP-trap resin and both inputs and captured fractions were examined by western blotting with antisera raised against phosphorylated CDK1 substrates and GFP (N = 3). ( F-H ) CHMP7-depleted HeLa cells stably expressing GFP-CHMP7 R , GFP-CHMP7 R S3A/S441A, or GFP-CHMP7 R S3D/S441D were imaged live during mitotic exit (H). In F, the fold-enrichment of GFP-signal at the reforming nuclear envelope was calculated from three independent experiments, presented as mean (N = 3) ± S.E.M. (WT, n = 24; S3A/S441A, n = 27, n.s. (p = 0.92), S3D/S441D, n = 52, p = 0.049; one-way ANOVA with Dunnett’s multiple comparisons). In G, the degree of furrow ingression (midzone diameter/daughter cell diameter) at the point of maximal GFP-CHMP7 recruitment was used as a proxy of progression through M-exit and was calculated from three independent experiments, presented as mean (N = 3) ± S.E.M. (WT, n = 24; S3A/S441A, n = 24, n.s. (p = 0.99), S3D/S441D, n = 46, p = 0.042; one-way ANOVA with Dunnett’s multiple comparisons). In H, for cells stably expressing GFP-CHMP7 R S3A/S441A, arrowheads depict precocious clustering of CHMP7 in the peripheral ER during anaphase that develop into larger clusters during mitotic exit. In all panels, time in minutes and a scale bar of 10 μm are reported. Figure 5—source data 1. Dynamics of CHMP7 assembly at the reforming nuclear envelope.

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Stable Transfection, Expressing, Inhibition, De-Phosphorylation Assay, SDS Page, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.7554/eLife.59999

Figure Lengend Snippet: ( A ) HeLa cells stably expressing either GFP-CHMP7 R or GFP-CHMP7 R S3A/S441A were imaged during mitosis. Arrowheads depict precocious assembly of GFP-CHMP7 R S3A/S441A in the peripheral ER. ( B, C ) Endogenous CHMP7 was depleted from HeLa cells stably expressing GFP-CHMP7 R S3A/S441A. Cells were either fixed and stained with antisera raised against IST1 or CHMP4B and the number of cells exhibiting ESCRT-III puncta were quantified ( B . Interphase; N = 4, n = 382 cells (GFP-CHMP7 R ); N = 4, n = 697 cells (GFP-CHMP7 R S3A/S441A); p = 0.002, two-tailed T-test. Mitosis, N = 3, n = 94 (GFP-CHMP7 R ); N = 4, n = 80 (GFP-CHMP7 R S3A/S441A); p = 0.03, two-tailed T-test. Data presented as mean ± S.D.). Figure 5—figure supplement 3—source data 1. CDK1 phosphorylation of CHMP7 suppresses formation of clusters of CHMP7 that grow during M-exit.

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Stable Transfection, Expressing, Staining, Two Tailed Test, Phospho-proteomics

Journal: eLife

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.7554/eLife.59999

Figure Lengend Snippet:

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Cloning, Transfection, Construct, Retroviral, Plasmid Preparation, Expressing, Modification, Sequencing, Produced, Recombinant, Protein Purification, Control

Journal: Journal of Virology

Article Title: The Severe Fever with Thrombocytopenia Syndrome Virus NSs Protein Interacts with CDK1 To Induce G 2 Cell Cycle Arrest and Positively Regulate Viral Replication

doi: 10.1128/jvi.01575-19

Figure Lengend Snippet: Fig. 5. NSs interacts with CDK1 and inhibits the cyclin B1-CDK1 complex from 744

Article Snippet: Primary 426 antibodies to CDK1 (cat. no. 9611s) and Cyclin B1 (cat. no. 9915s) were 427 purchased from Cell Signaling Technology.

Techniques:

Journal: Journal of Virology

Article Title: The Severe Fever with Thrombocytopenia Syndrome Virus NSs Protein Interacts with CDK1 To Induce G 2 Cell Cycle Arrest and Positively Regulate Viral Replication

doi: 10.1128/jvi.01575-19

Figure Lengend Snippet: Fig. 6. The interaction between CDK1 and NSs is IB-dependent. (A) Schematic of 770

Article Snippet: Primary 426 antibodies to CDK1 (cat. no. 9611s) and Cyclin B1 (cat. no. 9915s) were 427 purchased from Cell Signaling Technology.

Techniques:

Journal: Oncotarget

Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1

doi: 10.18632/oncotarget.23329

Figure Lengend Snippet: Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle (DMSO) as control and the other LB-100 (10 µM). Cells were then taken at the indicated time points of further incubation. Two cell samples in the control set also received again nocodazole (Noco) and one of them LB-100 (LB-100) and, then, were taken at the indicated time points of further incubation. ( A ) Total samples were separated on SDS/PAGE and immunoblotted for phosphorylated Cdk1 substrates (Cdk1 p-subs), pT481-PRC1, PRC1, cyclin B1 (Cyc B1) and Cdk1. ( B ) Optical pT481-PRC1 (triangles) and cyclin B1 (squares) signal density (arbitrary units; normalized for total PRC1 and Cdk1 optical density values, respectively) were plotted as percent of time 0 samples from control (open symbols) and LB-100-treated (filled symbols) cells. ( C ) Nocodazole-treated, prometaphase-arrested, hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (Noco– LB-100+) or fresh medium plus nocodazole and LB-100 (Noco+ LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). ( D ) Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus 10 µM LB-100 (Noco– LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments performed under identical conditions and giving similar results.

Article Snippet: Rabbit monoclonal anti-phospho-serine Cdk1 substrates (Cdk1 p-subs; recognizing PXpSP and pSPXK/R; 1:1000) and rabbit polyclonal anti phospho-pT320-PP1α (1:750) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) while mouse monoclonal anti-BubR1 (1:1000) antibody was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Control, Incubation, SDS Page