Journal: PLOS ONE

Article Title: Pathogen and human NDPK-proteins promote AML cell survival via monocyte NLRP3-inflammasome activation

doi: 10.1371/journal.pone.0288162

Figure Lengend Snippet: (A) Whole blood from normal donors was diluted and incubated with fluorescently labelled BSA control (in blue), rNM23-H1 (in pink) and S.pneumoniae rNDPKR (in grey). Representative plots for B-cells (CD19), T-cells (CD3), neutrophils (CD11b+CD14-) and monocytes (CD11b+CD14+) are shown (See for gating strategy). (B) Fluorescence geometric mean fold changes compared to BSA control of each individual cell type for n = 4 normal donors. (C) ELISA for IL-1β and IL-6 cytokines in conditioned media (CM) generated incubating for 18 hours diluted whole blood (closed circles) or PBMC (open squares) with rNM23-H1 or rNDPK from S.pneumoniae and C.albicans. (D) Red cells from normal donor’s whole blood were lysed and leukocytes were depleted from CD14+ cells (monocytes). Either total white cells or monocyte depleted cells were treated with NM23-H1 or rNDPK for 18h and IL-1β and IL-6 were analyzed by ELISA.

Article Snippet: Red cells were then lysed with red cell lysis buffer (155mM ammonium chloride, 10mM potassium bicarbonate, 0.1mM EDTA, pH 8.0) and leukocytes immunostained for CD19 (clone SJ25C1) CD3 (clone SK7), CD11b (clone D12), and CD14 (clone M5E2) (all BD Pharmingen), in the presence of 1:50 FcR Block (Miltenyi Biotec) before flow cytometric analysis.

Techniques: Incubation, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Generated

Journal: The Journal of Cell Biology

Article Title: Regulation of the Cell Cycle by Focal Adhesion Kinase

doi:

Figure Lengend Snippet: Regulation of the expression levels of components of G1/S cyclin-Cdk complexes by exogenous FAK and mutants. ( A ) α-p21, α-cyclin D1, α-Cdk2, α-cyclin E, α-cyclin A, or α-p27 Western blot of whole cell lysates prepared from cells expressing the indicated proteins under uninduced (lanes U ) and induced (lanes I ) conditions. ( B ) Serum-starved cells expressing ΔC14 were incubated in media containing 10% CS in the presence ( U ) or absence ( I ) of tetracycline. At the indicated times, lysates were prepared and analyzed by Western blot with α-p21, α-cyclin D1, α-Cdk2, α-cyclin E, or α-cyclin A.

Article Snippet: The following antibodies were purchased as indicated: α-BrdU mouse mAb from Sigma (St. Louis, MO); α-phosphotyrosine mouse mAb PY20 from Transduction Laboratories (Lexington, KY); rabbit α-Src, α-Fyn, α-Erk, α-cyclin A, α-cyclin E, α-Cdk2, α-His (His-Probe), and mouse mAb α-Shc from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit α-Shc from UBI (Lake Placid, NY); and rabbit α-phospho-MAPK from New England Biolabs, Inc. (Beverly, MA).

Techniques: Expressing, Western Blot, Incubation

Journal:

Article Title: Spy1 Interacts with p27 Kip1 to Allow G 1 /S Progression

doi: 10.1091/mbc.E02-12-0820

Figure Lengend Snippet: Human p27 interacts with human Spy1 in vitro. (A) In vitro–translated p27 (TNT-p27) was incubated for 2 h with either in vitro–translated pCS3-MT empty vector (TNT-mock) or in vitro–translated myc-tagged Spy1 (TNT-Spy1). The top panel demonstrates the amount of input TNT-Spy1 protein (lane 2), the middle panel demonstrates the input TNT-p27 protein (lanes 1 and 2). Both samples were immunoprecipitated with α-myc sera, and proteins were separated by 10% SDS-PAGE and immunoblotted with p27 sera (bottom panel). (B) In vitro–translated pCS3-MT (TNT-mock), p27 (TNT-p27), myc-Spy1 (TNT-Spy1), or CDK2 (TNT-CDK2) were separated by 10% SDS-PAGE and immunoblotted with α-CDK2 sera as a control. (C) Purified GST-p27 or GST-control protein conjugated to GST beads were incubated in binding buffer with either in vitro–translated (TNT) mock or Spy1 protein (top panel), both of which were previously immunodepleted for any CDK2 protein as an additional control. A GST pull-down assay was performed. Proteins were separated by 12.5% SDS-PAGE and immunoblotted with α-myc sera to detect immunoprecipitation of GST proteins with myc-tagged proteins (bottom panel). α-GST immunoblot is presented to demonstrate the GST proteins present in the GST pull-down experiment. Different exposures of the same immunoblot are presented in the middle panel because of the high amount of protein in the GST-control lanes.

Article Snippet: Lysates (1 mg protein) were precleared with 40 μl Protein A-Sepharose beads and incubated with 2 μg of α-flag (Sigma), α-myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), α-p27 (Zymed, South San Francisco, CA, or Santa Cruz) or α-CDK2 (Santa Cruz) overnight at 4°C.

Techniques: In Vitro, Incubation, Plasmid Preparation, Immunoprecipitation, SDS Page, Purification, Binding Assay, Pull Down Assay, Western Blot

Journal:

Article Title: Spy1 Interacts with p27 Kip1 to Allow G 1 /S Progression

doi: 10.1091/mbc.E02-12-0820

Figure Lengend Snippet: p27 interacts with Spy1 and CDK2. 293T cells were transfected with the indicated constructs. The lysates were immunoprecipitated with α-p27 or α–CDK2 sera and immunoblotted for Spy1, CDK2, and p27. Lane 8 demonstrates that flag-Spy1 (top panel), endogenous CDK2 (middle panel), and p27 (bottom panel) can coimmunoprecipitate. The top panel of lane 11 shows that Spy1 interacts with CDK2 and that this interaction is increased when p27 is overexpressed as demonstrated in lane 12.

Article Snippet: Lysates (1 mg protein) were precleared with 40 μl Protein A-Sepharose beads and incubated with 2 μg of α-flag (Sigma), α-myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), α-p27 (Zymed, South San Francisco, CA, or Santa Cruz) or α-CDK2 (Santa Cruz) overnight at 4°C.

Techniques: Transfection, Construct, Immunoprecipitation

Journal:

Article Title: Spy1 Interacts with p27 Kip1 to Allow G 1 /S Progression

doi: 10.1091/mbc.E02-12-0820

Figure Lengend Snippet: Spy1 enhances histone H1 kinase activity in the presence of p27. (A) 293T cells were transfected with the indicated constructs. The lysates were immunoprecipitated with α-CDK2 sera and subjected to an in vitro kinase assay. Lane 2 shows that p27-transfected cells have decreased kinase activity, but Spy1-transfected cells exhibit kinase activity higher than mock-transfected cells (lane 3). When both Spy1 and p27 are cotransfected, CDK2 histone H1 kinase activity is increased as shown in lane 4 compared with p27 alone. (B) The lysates were analyzed by 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with flag-Spy1, CDK2, and p27 antisera to demonstrate equivalent protein expression.

Article Snippet: Lysates (1 mg protein) were precleared with 40 μl Protein A-Sepharose beads and incubated with 2 μg of α-flag (Sigma), α-myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), α-p27 (Zymed, South San Francisco, CA, or Santa Cruz) or α-CDK2 (Santa Cruz) overnight at 4°C.

Techniques: Activity Assay, Transfection, Construct, Immunoprecipitation, In Vitro, Kinase Assay, SDS Page, Expressing

Journal:

Article Title: Spy1 Interacts with p27 Kip1 to Allow G 1 /S Progression

doi: 10.1091/mbc.E02-12-0820

Figure Lengend Snippet: Spy1-enhanced proliferation is dependent on endogenous p27. (A) MEF–/– cells were transfected with the indicated constructs, medium was changed 24 h posttransfection, and cells were counted via trypan blue exclusion 48 h posttransfection. The graphed data indicate that exogenous p27 slows proliferation in the MEF–/– cells and that Spy1 can overcome this inhibition of proliferation. Spy1 does not enhance proliferation over mock control in cells lacking endogenous p27. The data are one representative experiment of four. Error bars indicate the SEM of three separate transfections. (B) Lysates were analyzed by 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with α-myc-Spy1, and α-p27 sera to demonstrate protein expression. (C) Endogenous Spy1 was immunoprecipitated from either NIH3T3 cells null for p27 (3T3–/–) or NIH3T3 wt (3T3wt). Both lysate and immunoprecipitated samples were separated by 10% SDS-PAGE and immunoblotted with α-CDK2 sera (top panel) or α-p27 sera (bottom panel). (D) Endogenous CDK2 was immunoprecipitated from both 3T3wt and 3T3–/– cell types. Lysate samples and the immunoprecipitated samples were analyzed by 10% SDS-PAGE, and immunoblotting was carried out with α-Spy1 sera.

Article Snippet: Lysates (1 mg protein) were precleared with 40 μl Protein A-Sepharose beads and incubated with 2 μg of α-flag (Sigma), α-myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), α-p27 (Zymed, South San Francisco, CA, or Santa Cruz) or α-CDK2 (Santa Cruz) overnight at 4°C.

Techniques: Transfection, Construct, Inhibition, SDS Page, Expressing, Immunoprecipitation, Western Blot