Journal: PLoS Pathogens

Article Title: SAMHD1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity

doi: 10.1371/journal.ppat.1008855

Figure Lengend Snippet: a) HFFs were pretreated for 5 h with the Cdk1 inhibitor CGP74514A (CGP), and then not infected or infected with AD169, in the presence of CGP. After removing virus inoculum, the inhibitor was newly added and kept until cell harvesting, at 3 dpi. Total or pT592-SAMHD1 levels were analyzed by immunoblotting, together with IE1/IE2 and p85 expression. A representative experiment out of five is shown. b) The relative amount of pT592-SAMHD1, normalized to that of p85, was determined by densitometric analysis and is relative to that of n.i. cells, which was arbitrarily set as 1. Data are expressed as mean ± SE of four independent experiments performed at 1 and 3 dpi. c) Effect of CGP on infectious virus production as measured by standard plaque assays on cell culture supernatants at 3 dpi. Results derive from seven experiments and are expressed as mean ± SE. d) HFFs were pretreated as in panel a) with the Cdk inhibitors RO-3306 (RO), KO-3861 (KO) or BMS-265246 (BMS) at the indicated concentrations (μM), and then not infected or infected with AD169, in the presence of the inhibitors. After removing virus inoculum, the drugs were newly added and kept until cell harvesting, at 1 dpi. Immunoblotting was performed as above. One representative experiment out of two is shown. e) HFFs were transfected with Cdk1 or Cdk2 siRNA or non-targeting siRNA (ctrl). The day after, cells were not infected or infected with AD169 at an MOI of 1 and harvested at 1 dpi. Immunoblotting was performed as above. One representative experiment out of two is shown. NT, not treated cells; DMSO, cells treated with the vehicle DMSO; *, unspecific band; black arrow, pT592-SAMHD1 specific band. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; *****, p< 0.00001.

Article Snippet: The small interfering RNA (siRNA) specific for SAMHD1 (sc-76442), for Cdk1 (sc-29252), Cdk2 (sc-29259) and the non-targeting siRNA (siCtrl) (sc-37007) were from Santa Cruz Biotechnology.

Techniques: Infection, Virus, Cell Harvesting, Western Blot, Expressing, Cell Culture, Transfection

Journal:

Article Title: BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

doi:

Figure Lengend Snippet: BRCA1 is phosphorylated by CDK2-cyclin complexes in vitro. (A) Myc-BRCA1 wt was transiently transfected into 293T cells and immunoprecipitated with antibody 9E10. The immunoprecipitate was phosphorylated in vitro in the absence (lane 1) or presence of a recombinant CDK-cyclin complex: CDK2-cyclin E (lane 2), CDK6-cyclin D1 (lane 3), CDK2-cyclin A (lane 4), or CDC2-cyclin B1 (lane 5). The reaction products were visualized by autoradiography following SDS-polyacrylamide gel electrophoresis and transfer to a membrane. The 200-kDa protein marker is indicated on the left. (B) Two-dimensional tryptic phosphopeptide maps derived from BRCA1 protein depicted in panel A. a, b, and c, maps of BRCA1 from lanes 1, 3, and 5, respectively. Phosphopeptides p4, pA, and pB are depicted in Fig. ​Fig.44B.

Article Snippet: The following antibodies were used: BRCA1 Ab-D ( 57 ), Myc-specific antibody 9E10 (Jill Meisenhelder, The Salk Institute), and mouse CDK2-specific monoclonal antibody (D-12; Santa Cruz).

Techniques: In Vitro, Transfection, Immunoprecipitation, Recombinant, Autoradiography, Polyacrylamide Gel Electrophoresis, Marker, Derivative Assay

Journal:

Article Title: BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

doi:

Figure Lengend Snippet: An endogenous protein kinase activity as well as recombinant CDK2-cyclin complexes phosphorylate BRCA1 residues T967 and S1497 in vitro. (A) 293T cells were transiently transfected with Myc-BRCA1 that was either wild type (Wt; lanes 1 and 2) or mutated as indicated. The different BRCA1 species were immunoprecipitated with antibody 9E10 and subjected to in vitro kinase reactions in the absence (lanes 1, 3, 5, 7, and 9) or presence (lanes 2, 4, 6, 8, and 10) of the recombinant CDK2-cyclin A complex. The 200-kDa protein marker is shown on the left. (B) Two-dimensional tryptic phosphopeptide maps derived from the overexpressed BRCA1 protein species depicted in panel A. a to j, maps of BRCA1 from lanes 1, 3, 5, 2, 4, 6, 7, 9, 8, and 10, respectively. (C) Two-dimensional maps of mixtures of tryptic phosphopeptides derived from in vitro-phosphorylated and in vivo-labeled BRCA1. a, in vitro-phosphorylated BRCA1 alone; b, in vivo-labeled endogenous BRCA1 from HBL-100 cells alone; c, mixture of in vitro-phosphorylated BRCA1 and in vivo-labeled endogenous BRCA1 from HBL-100 cells; d, in vivo-labeled overexpressed BRCA1 in 293T cells alone; e, mixture of in vitro-phosphorylated BRCA1 and in vivo-labeled overexpressed BRCA1 in 293T cells. Numbered circles indicate phosphopeptides p3, p4, p9, p11, p18, p20, pA, and pB (for simplicity, only a few phosphopeptides were circled); unlabeled circles indicate the lack of the respective peptides; circles labeled by an asterisk mark phosphopeptides related to pA (see text); T (threonine) and S (serine) indicate the phosphoamino acid content of the corresponding phosphopeptide.

Article Snippet: The following antibodies were used: BRCA1 Ab-D ( 57 ), Myc-specific antibody 9E10 (Jill Meisenhelder, The Salk Institute), and mouse CDK2-specific monoclonal antibody (D-12; Santa Cruz).

Techniques: Activity Assay, Recombinant, In Vitro, Transfection, Immunoprecipitation, Marker, Derivative Assay, In Vivo, Labeling

Journal:

Article Title: BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

doi:

Figure Lengend Snippet: BRCA1 coimmunoprecipitates with CDK2 and cyclin A. (A) Myc-BRCA1 wt coimmunoprecipitates with CDK2. 293T cells were transfected with expression plasmids for the following proteins: lane 1, Myc-BRCA1 wt; lane 2, Myc-BRCA1 wt, cyclin A, and CDK2; lane 3, Myc-BRCA1 wt, cyclin E, and CDK2; lane 4, cyclin A and CDK2; lane 5, cyclin E and CDK2; lane 6, Myc-BRCA1 wt and CDK2; and lane 7, CDK2. CDK2-containing complexes were immunoprecipitated (I.P.) from each sample with a rabbit CDK2 antibody (α-CDK2) and analyzed after separation on SDS-polyacrylamide gels by Western blot analysis for the presence of Myc-BRCA1 wt, using antibody 9E10 (a, bottom row), and CDK2, using a mouse anti-CDK2 monoclonal antibody (b, bottom row). The two top rows in panels a and b (Input) represent the relative amounts of Myc-BRCA1 wt and CDK2 in the cell lysates before immunoprecipitation. Panel c shows that in a duplicate experiment, Myc-BRCA1 wt from cell lysates overexpressing proteins as indicated in lanes 1, 2, 3, and 6 (in panels, a and b) was further resolved on an SDS-polyacrylamide gel (compared to panel a, top row) in order to increase resolution. (B) Untagged, full-length wild-type BRCA1 coimmunoprecipitates with CDK2 and cyclin A. Lysates from a HeLa cell line stably overexpressing BRCA1 (57a) were subjected to immunoprecipitations using the following antibodies: lane 1, an aliquot before immunoprecipitation; lane 2, anti-CDK2; lane 3, anti-CDK2, after preincubation with the blocking peptide; lane 4, anti-NF-κB p65 as a control; lane 5, anti-cyclin A; and lane 6, anti-cyclin E. Western blot analysis revealed the presence of BRCA1 and CDK2, as shown in the upper and lower panels, respectively. The 250- and 42-kDa markers are indicated on the left.

Article Snippet: The following antibodies were used: BRCA1 Ab-D ( 57 ), Myc-specific antibody 9E10 (Jill Meisenhelder, The Salk Institute), and mouse CDK2-specific monoclonal antibody (D-12; Santa Cruz).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Stable Transfection, Blocking Assay

Journal:

Article Title: BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

doi:

Figure Lengend Snippet: Phosphorylation of BRCA1 is decreased by inhibition of CDK2. (A) CDK2 dn decreases the phosphorylation state of endogenous BRCA1. 293T cells were transiently transfected with a control plasmid (lane 1) or a plasmid expressing CDK2 dn (lane 2). Endogenous BRCA1 from both sources was immunoprecipitated with Ab-D prior to treatment with (lanes 4 and 6) or without (lanes 3 and 5) BAP before analysis by immunoblotting with Ab-D. Filled arrow, BRCA1 from mock-transfected cells; open arrow, faster-migrating BRCA1 species due to expression of CDK2 dn; asterisk, BAP-treated BRCA1. (B) BRCA1 is phosphorylated in vitro by a cellular kinase activity that is sensitive to CDK2 dn. 293T cells were cotransfected with a plasmid expressing Myc-BRCA1 wt and either a control plasmid (lane a) or CDK2 dn (lane b). BRCA1 was immunoprecipitated under native conditions with antibody 9E10, and in vitro kinase reactions were performed on the immunoprecipitates. a and b, two-dimensional tryptic phosphopeptide maps of the BRCA1 protein species shown in lanes a and b, respectively, from a duplicate experiment. (C) p21 and butyrolactone I but not p16 inhibit the endogenous protein kinase activity that phosphorylates BRCA1 at T967 and S1497 in vitro. Myc-BRCA1 wt was subjected to in vitro kinase reactions as described above. Lane a, no CDI added; lanes b, c, and d, p16, p21, and butyrolactone I (Butyr), respectively, added. a to d, phosphopeptide maps of BRCA1 protein shown in lanes a to d, respectively. The top portions of panels B and C represent the amount of (Western blot analysis using antibody 9E10 [bottom rows]) and the in vitro kinase activity on (radioactive incorporation onto BRCA1 [top rows]) BRCA1. The 250- and 200-kDa markers are shown on the left.

Article Snippet: The following antibodies were used: BRCA1 Ab-D ( 57 ), Myc-specific antibody 9E10 (Jill Meisenhelder, The Salk Institute), and mouse CDK2-specific monoclonal antibody (D-12; Santa Cruz).

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, In Vitro, Activity Assay

Journal: Molecular Biology of the Cell

Article Title: miR-29b represses intestinal mucosal growth by inhibiting translation of cyclin-dependent kinase 2

doi: 10.1091/mbc.e13-05-0287

Figure Lengend Snippet: FIGURE 4: miR-29b associates with the cdk2 mRNA. (A) Schematic representation of cdk2 mRNA depicting predicted target site for miR-29b in its 3′-UTR. (B) Levels of biotinylated miR-29b were measured by Q-PCR analysis 24 h after transfection. Values are means ± SEM from three separate experiments. *p < 0.05 compared with scramble. (C) Binding of biotinylated miR-29b to cdk2, cccnd1, xiap, and cdk4 mRNAs in the materials pulled down by biotin–miR-29b.

Article Snippet: The expression vector containing DDK-tagged CDK2 protein was obtained from OriGene (Rockville, MD).

Techniques: Transfection, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: miR-29b represses intestinal mucosal growth by inhibiting translation of cyclin-dependent kinase 2

doi: 10.1091/mbc.e13-05-0287

Figure Lengend Snippet: FIGURE 5: miR-29b inhibits CDK2 translation. (A) Levels of miR-29b 48 h after transfection of pre–miR-29b. Values are means ± SEM from three separate experiments. *p < 0.05 compared with scramble. (B) The levels of CDK2 protein (a) and its new synthesis (b) after ectopic miR-29b overexpression. After cells were exposed to l-azidohomoalanine (AHA), cell lysates were incubated with the reaction buffer containing biotin/alkyne reagent; the biotin-alkyne/azide– modified protein complex was pulled down by paramagnetic streptavidin-conjugated Dynabeads. (C) Polysomal profiles in cells described in A. Nuclei were pelleted, and the resulting supernatants were fractionated through a 10–50% linear sucrose gradient. (D) Distributions of cdk2 (top) and gapdh (bottom) mRNAs in each gradient fraction prepared from cells described in C. The levels of cdk2 and gapdh mRNAs were plotted as a percentage of the total cdk2 or gapdh mRNA levels in the samples. (E) Levels of reporter activities after ectopic overexpression of miR-29b. Results are expressed as the means ± SEM data from three separate experiments.

Article Snippet: The expression vector containing DDK-tagged CDK2 protein was obtained from OriGene (Rockville, MD).

Techniques: Transfection, Over Expression, Incubation, Modification

Journal: Molecular Biology of the Cell

Article Title: miR-29b represses intestinal mucosal growth by inhibiting translation of cyclin-dependent kinase 2

doi: 10.1091/mbc.e13-05-0287

Figure Lengend Snippet: FIGURE 6: Effect of miR-29b–regulated CDK2 expression on cell proliferation. (A) Changes in cell growth after ectopic overexpression of miR-29b. Values are means ± SEM from three separate experiments. *p < 0.05 compared with cells transfected with scramble. (B) Flow cytometric analysis of cell cycle distribution after miR-29b overexpression for 72 h. (C) The relative G1, S, and G2/M compartments calculated from data described in B. (D, E) Changes in levels of CDK2 and cell growth after cotransfection the pre–miR-29b and CDK2 expression vector containing DDK-tagged CDK2 (DDK-CDK2). After cells were transfected with either control scramble or premiR-29b for 24 h, they were transfected with the CDK2 expression vector or control vector (Null). The levels of CDK2 protein (D) and cell growth (E) were examined 48 h after CDK2 overexpression. *,+p < 0.05 compared with scramble and pre–miR-29b, respectively.

Article Snippet: The expression vector containing DDK-tagged CDK2 protein was obtained from OriGene (Rockville, MD).

Techniques: Expressing, Over Expression, Transfection, Cotransfection, Plasmid Preparation, Control