Journal: PLoS ONE
Article Title: Cell Cycle Regulated Phosphorylation of the Telomere-Associated Protein TIN2
Figure Lengend Snippet: TIN2 is phosphorylated by the mitotic kinase RSK2. ( A ) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. ( B ) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. ( C ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. ( D ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2 Y707A ) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left ( top ). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control ( bottom ). Representative of two experiments. ( E ) Detection of TIN2 phosphorylation by RSK2 in vitro . Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP 32 , after which the reaction products were resolved by SDS-PAGE and either ( top ) exposed to autographic film or ( bottom ) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P 32 ) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.
Article Snippet: Cell Synchronization by Nocodazole Arrest and Double Thymidine Block 106 of the described stably infected HeLa cells were either left untreated, arrested in G2/M by overnight treatment with 0.6 µg/ml nocodazole (catalogue # M1404, Sigma), or synchronized at G1/S by the double thymidine block, as outlined below.
Techniques: Stable Transfection, Expressing, Derivative Assay, Immunoprecipitation, SDS Page, Staining, Fluorescence, FACS, Transfection, Mutagenesis, Activity Assay, In Vitro, Recombinant, Binding Assay, Purification, Incubation