Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

Article Snippet: The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), according to the manufacturers’ instructions.

Techniques: Blocking Assay, Sample Prep, Cytometry, Microscopy, Western Blot

Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

Article Snippet: The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), according to the manufacturers’ instructions.

Techniques: Expressing, Blocking Assay, Immunoprecipitation, Western Blot, Purification, In Vitro, Kinase Assay

Journal: The Journal of Cell Biology

Article Title: The ubiquitin ligase CRL2 ZYG11 targets cyclin B1 for degradation in a conserved pathway that facilitates mitotic slippage

doi: 10.1083/jcb.201601083

Figure Lengend Snippet: Human CRL2 ZYG11B interacts with cyclin B1 independently of Cdk1. (A) FLAG-ZYG11B interacts with Venus–cyclin B1 mutant proteins that are incapable of binding Cdk1. FLAG-ZYG11B was coexpressed with wild-type or mutated versions of Venus–cyclin B1 with R202A or R202A R42A substitutions in HEK293T cells that were treated with MG132. The Venus tag was immunoprecipitated and immunoblotted for Venus–cyclin B1, FLAG-ZYG11B, or Cdk1 (left). Similar results were obtained in two independent experiments. (B) Endogenous Cdk1 coprecipitates HA-ZYG11B but not control HA–leucine-rich repeat (LRR) 1 in HEK293T cells treated with MG132. Similar results were obtained in two independent experiments. (C) HA-ZYG11B predominantly interacts with the CBOX1 domain of cyclin B1. HA-ZYG11B was coexpressed with Venus–cyclin B1, Venus-tagged truncations of cyclin B1 (N terminus, CBOX1, or CBOX2), or pEGFP-N1 control vector in HEK293T cells treated with MG132. Note that the truncations were cleaved into smaller isoforms in vivo (the full-length size is marked by an arrow on the right side). The Venus tag was immunoprecipitated and immunoblotted for HA or Venus tags (left panels). The results are representative of five independent experiments.

Article Snippet: ZYG11B was cloned into pEGFP-N1, after the EGFP sequence was removed, as an N-terminal HA-tagged protein. pVenus-N1 cyclin B1 and pVenus–cyclin B1 (R42A) expression vectors were created by the Pines laboratory ( ) and obtained from Addgene (plasmids 26062 and 39873, respectively). pVenus-N1 cyclin B1 (R202A and R42A) was made by introducing the R202A mutation into the pVenus-N1 cyclin B1 (R42A) construct using an overlap-extension mutagenesis method ( ).

Techniques: Mutagenesis, Binding Assay, Immunoprecipitation, Control, Plasmid Preparation, In Vivo