m tris hcl buffer  (Millipore)

Journal: Cell Reports

Article Title: Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1

doi: 10.1016/j.celrep.2018.12.082

Figure Lengend Snippet: Mutational Analysis of the DNA-Binding Site (A) Solvent-accessible surface of the DNA-binding site colored by electrostatic potential (calculated in PyMol). Residues perturbed by DNA binding (including Arg399 and Arg400) map to an intensely positively charged surface patch (left), whose polarity is predicted to be reversed by the combination of R399D and R400Q mutations (right). ) of XRCC1-BRCT1 variants and mutants. Both codon 399 variants and the putative DNA binding disruptive R399D,R400Q double mutant bind tightly to PAR chains generated on plates coated with histone H1 and incubated with PARP1 and NAD + ). No binding is seen for any of the constructs in the absence of NAD + . Data represent the mean of four measurements of three separate replicates analyzed by two-way ANOVA. Error bars show ± 1 SEM. (C) Fluorescence polarization assays of XRCC1-BRCT1 variants and mutants to FITC-labeled nicked dsDNA oligonucleotides with (left) or without (right) 5′ phosphorylation at the nick site. The codon 399 variants and the PAR-binding site mutant all bind with high affinity to both nicked duplex oligonucleotides, whereas the R399D,R400Q double mutant shows very low fluorescence poloarization (FP) values, which cannot be fitted to a binding curve (for K d B). Data represent the mean of four measurements comprised of two separate replicates with XRCC1-BRCT1 from two separate protein purifications. Error bars show ± 1 SEM.

Article Snippet: The adsorbed proteins were mock ribosylated in the absence of NAD+ or ribosylated in the presence of the 50 mM NAD+ (Sigma) in PARP1 reaction buffer (50 mM Tris–HCl pH7.5, 0.8 mM MgCl2 , 1% glycerol and 1.5 mM DTT) containing 40 nM single-stranded oligodeoxyribonucleotide (5′-CATATGCCGGAGATCCGCCTCC-3′) and 5 nM human PARP1 (Trevigen) in a final volume of 50 μL at room temp for 30 min. After rinsing (4 × ) with 50 μL of 0.1% Tween 20 in PBS, 50 μL of His-SUMO-XRCC-BRCT1 or its variants (diluted to 25 nM in 20 mM Tris pH7.5, 130 nM NaCl) were added to the adsorbed proteins and incubated on ice for 30 min.

Techniques: Binding Assay, Mutagenesis, Generated, Incubation, Construct, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Regulation by the Wilms Tumor Protein, Wt1, Suggests a Role of the Metalloproteinase Adamts16 in Murine Genitourinary Development *

doi: 10.1074/jbc.M113.464644

Figure Lengend Snippet: Binding of Wt1 protein to the 5′-flanking region of the Adamts16 gene. ChIP was performed to detect Wt1 protein bound to the 5′-flanking region of the Adamts16 gene in its native chromosomal configuration. The drawing ( A ) delineates the three predicted Wt1 binding sites ( Wt1-A , Wt1-B , and Wt1-C ) in the promoter and 5′-UTR of the Adamts16 gene and allocates the PCR primers used for DNA amplification. Specific antibodies against Wt1 and histone proteins were chosen for immunoprecipitation of M15 whole cell lysates. Amplicons encompassing the 5′-flanking region of the Adamts16 gene were enriched ∼2.5-fold with the use of anti-Wt1 antibody compared with normal rabbit IgG (NRb-IgG). No differences in actin DNA were observed between anti-Wt1 antibody and normal rabbit IgG ( B ). The gene encoding anti-Müllerian hormone receptor 2 ( Amhr2 ), served as a positive control ( B ). Binding of Wt1(−KTS) protein to the Adamts16 promoter was confirmed in stimulated UB27 cells ( C ). Wt1(+KTS) protein failed to interact with the promoter of the Adamts16 gene in UD28 cells ( D ). Data shown are means ± S.E. ( error bars ). Statistical significances are indicated by asterisks (*, p

Article Snippet: The supernatants were diluted in immunoprecipitation buffer (0.01% SDS, 1.1% Triton X-100, 1.2 m m EDTA, 16.7 m m Tris-HCl, pH 8.1) and precleared for 1 h at 4 °C with DNA-blocked protein G-agarose (Millipore).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Positive Control

Journal: Journal of Virology

Article Title: Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus ▿

doi: 10.1128/JVI.01306-07

Figure Lengend Snippet: Effects of NEM on the UL16-capsid interaction. (A) Extracellular virions were treated with or without NEM at 37°C. Viral membranes were removed with NP-40 treatment, and the capsids were pelleted through a 30% sucrose cushion prior to immunoblot analyses with antibodies specific for VP5, VP16, and UL16. (B) Extracellular virions were treated with or without NEM for 30 min and then treated with or without NP-40. The released capsids or intact virions (C or V, respectively) were pelleted through a 30% sucrose cushion prior to immunoblotting with the indicated antiserum. (C) Virions present in the medium were concentrated by centrifugation through a sucrose cushion and resuspended in a small volume of TNE buffer. The sample was divided into two equal portions, and these were treated with or without NEM for 30 min. Both samples then received NP-40 for 15 min at room temperature prior to sedimentation through parallel 20 to 50% sucrose gradients. Fractions were collected from the top and analyzed by immunoblotting with antibodies specific for VP5 and UL16. (D) Gradient-purified capsids treated with NEM or carrier prior to lysis for 15 min at 37°C from infected cytoplasm and medium were analyzed by immunoblotting for levels of VP5 and UL16. Gradients were run in duplicate. α, anti.

Article Snippet: At 16 to 20 h postinfection, cells were scraped into 20 ml of PBS, collected by centrifugation at 1,000 × g for 10 min, resuspended in 6 ml of NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 1 M Tris-HCl, pH 8.0) containing protease inhibitors (P8340; Sigma), and incubated for 15 min on ice.

Techniques: Centrifugation, Sedimentation, Purification, Lysis, Infection

Journal: Journal of Virology

Article Title: Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus ▿

doi: 10.1128/JVI.01306-07

Figure Lengend Snippet: Effects of low pH on the association of UL16 with capsids. Extracellular virions were harvested 20 to 24 h postinfection, pelleted through a 30% sucrose cushion, and resuspended in medium buffered to the indicated pH values. (A) After an incubation of 1 h at 37°C, the viral envelopes were removed with NP-40 treatment for 10 min at room temperature. The released capsids were pelleted, dissolved in sample buffer, and analyzed by immunoblotting with anti-VP5 and anti-UL16 antibodies. (B) Virions were first incubated at the indicated pH values for 30 min at 37°C and then incubated for an additional 30 min, either at the same pH or at pH 7.4 (following titration with NaOH). The viral envelopes were removed with NP-40, and the released capsids were collected and analyzed by immunoblotting. (C) Virions were treated as described for the previous panel except that the timing of the NP-40 treatment was either before or after neutralization with NaOH, as indicated by the asterisks. Relative levels of UL16 quantitated by densitometry and normalized for VP5 are indicated bellow each figure. α, anti.

Article Snippet: At 16 to 20 h postinfection, cells were scraped into 20 ml of PBS, collected by centrifugation at 1,000 × g for 10 min, resuspended in 6 ml of NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 1 M Tris-HCl, pH 8.0) containing protease inhibitors (P8340; Sigma), and incubated for 15 min on ice.

Techniques: Incubation, Titration, Neutralization

Journal: Journal of Virology

Article Title: Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus ▿

doi: 10.1128/JVI.01306-07

Figure Lengend Snippet: UL16-capsid interactions in virions. (A) Extracellular virions were harvested from HSV-infected Vero cells and pelleted through a 30% sucrose cushion. The samples were resuspended in TNE buffer, separated into six equal fractions, and treated as indicated for 15 min at room temperature. Following treatment, virions and capsids were pelleted through an additional sucrose cushion, resuspended in sample buffer, and separated by SDS-PAGE in 7% gels. Proteins were analyzed by immunoblotting using antibodies specific for VP5, VP16, and UL16. (B) Capsids were isolated from NP-40-treated cytoplasmic (cyt) lysates or extracellular medium by centrifugation through a 30% sucrose cushion. Capsid pellets were resuspended in sample buffer, and two different amounts were analyzed by immunoblotting using antibodies specific for VP5 and UL16. α, anti; Tryp, trypsin; Inh, inhibitors; Undil, undiluted.

Article Snippet: At 16 to 20 h postinfection, cells were scraped into 20 ml of PBS, collected by centrifugation at 1,000 × g for 10 min, resuspended in 6 ml of NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 1 M Tris-HCl, pH 8.0) containing protease inhibitors (P8340; Sigma), and incubated for 15 min on ice.

Techniques: Infection, SDS Page, Isolation, Centrifugation

Journal: The Journal of Biological Chemistry

Article Title: Palmitoylation of Desmoglein 2 Is a Regulator of Assembly Dynamics and Protein Turnover *

doi: 10.1074/jbc.M116.739458

Figure Lengend Snippet: Palmitoylation-deficient Dsg2 localizes to a perinuclear subcellular compartment. A–L , A431 cells stably expressing wild-type Dsg2/GFP ( A–C ), Dsg2/GFP 7mut ( D–F ), Dsg2/GFP 5mut ( G–I ), and Dsg2/GFP CACS ( J–L ) were immunostained using anti-desmoplakin antibody, and co-localization of GFP with desmoplakin is shown. The localization of Dsg2/GFP fusion proteins was determined in at least three independent cultures grown on individual coverslips. Scale bar = 10 μm. M , whole cell lysates were prepared from A431 cells expressing wild-type Dsg2/GFP and Dsg2/GFP CACS. Lysates were subjected to immunoblot ( IB ) analysis. Immunoblot analysis was performed using cell lysates prepared from three different cultures. These data demonstrate equal expression of the GFP fusion proteins. N , A431 cells expressing Dsg2/GFP and Dsg2/GFP CACS were separated into Triton X-100-insoluble ( P ) and Triton X-100-soluble ( S ) fractions and subjected to immunoblot analysis using anti-GFP. O , quantitation of soluble and insoluble fractions shown in N . The lysates were prepared from four independent cultures, and immunoblot analysis band intensities were collected using LiCor Odyssey infrared scanning. Student's t test was performed to determine differences in solubility (*, p

Article Snippet: Cells grown in a T25 flask were scraped into 1 ml of Triton X-100-containing buffer (10 m m Tris-HCl (pH 7.0), 0.5% Triton X-100 (Sigma), 5 m m EDTA, 2 m m EGTA, 30 m m sodium fluoride, 40 m m β-glycerophosphate, 10 m m sodium pyrophosphate, 2 m m sodium orthovanadate, and 2 m m phenylmethylsulfonyl fluoride) and incubated on ice while shaking for 15 min.

Techniques: Stable Transfection, Expressing, Quantitation Assay, Solubility