Journal: The Journal of Biological Chemistry

Article Title: Cellular requirements for iron–sulfur cluster insertion into the antiviral radical SAM protein viperin

doi: 10.1074/jbc.M117.780122

Figure Lengend Snippet: Model for the mode of interaction of CIA-targeting factors with viperin and for the unique maturation pathway. A, multimeric CIA-targeting complexes are present in the cell. As shown here, the CIA1–CIA2B–MMS19 complex interacts with the C terminus of viperin via direct contacts of both CIA1 and CIA2B. The two factors in turn recruit MMS19 to viperin. CIA2A binds at the N terminus of viperin and is stabilized by CIA1. Insertion of the radical SAM Fe/S cluster in the middle domain is mediated by C-terminally bound CIA1 only (see Ref. 6 and this study). B, Fe/S cluster maturation of viperin represents a novel branch of the CIA pathway that is initiated by the mitochondrial ISC machinery, early-acting CIA components, and IOP1 (20, 21). The components of the CIA1–CIA2B–MMS19-targeting complex operate in various combinations to facilitate Fe/S cluster insertion into dedicated client proteins, including GPAT, POLD1, and DPYD. CIA2A is specifically required for the maturation of IRP1 thus affecting cellular iron homeostasis and is stabilized by binding to CIA1. In contrast to all other known client Fe/S proteins, CIA1 appears to mediate Fe/S cluster insertion into the radical SAM protein viperin independently of the other CIA-targeting factors, thus representing a minimal requirement for CIA proteins.

Article Snippet: The rb α-FLAG epitope (F7425, d 1:5000), ms α-FLAG epitope (200472, d 1:2500), and rb α-POLD1 (15646–1-AP, d 1:1000) were obtained from Sigma, Stratagene, and Proteintech, respectively.

Techniques: Binding Assay

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