Journal: Plant Physiology

Article Title: The CPH1 Gene of Chlamydomonas reinhardtii Encodes Two Forms of Cryptochrome Whose Levels Are Controlled by Light-Induced Proteolysis 1 Encodes Two Forms of Cryptochrome Whose Levels Are Controlled by Light-Induced Proteolysis 1 [w]

doi: 10.1104/pp.103.031930

Figure Lengend Snippet: CPH1 migrates differently when overexpressed in E. coli or C. reinhardtii . A, E. coli cells transformed with pCPH101, which contains CPH1 cDNA, were induced with isopropylthio- β -galactoside for 2.5 h and cell extracts were analyzed using SDS-PAGE and stained with Coomassie Brilliant Blue. B, Western blot of E. coli pCPH101 extract before and after induction (left) and C. reinhardtii OxG extracts before and after heat shock (right). Western blots were performed using antibodies directed against CPH1 protein as described except TBST plus 0.3% (v/v) Triton X-100 was used.

Article Snippet: Primary antibodies against CPH1 were diluted to 1:1,000 in 1% (w/v) bovine serum albumin in TBST and incubated with the membrane for 1 h. M2 monoclonal antibodies against FLAG (Invitrogen, Carlsbad, CA) were diluted 1:1000 in 1% bovine serum albumin, 2.5% NFDM in TBST, and incubated with the membrane for 1 h. Regardless of the primary antibody used, membranes were subsequently washed for 30 min in TBST or TBST plus 0.3% (v/v) Triton X-100.

Techniques: Transformation Assay, SDS Page, Staining, Western Blot

Journal: Applied and Environmental Microbiology

Article Title: Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species

doi: 10.1128/AEM.03538-15

Figure Lengend Snippet: Concentration of RNA as a function of the A 260 / A 280 ratio of each of 180 RNA samples extracted from uninfected A. sativa (squares), K. macrantha (triangles), and A. gerardii (circles) plant tissues using Plant RNA Reagent (black), TRIzol (gray), and TRI

Article Snippet: Finally, we tested how efficiently each extraction reagent (TRIzol [Invitrogen], TRI Reagent [Sigma-Aldrich], and Plant RNA Reagent [Invitrogen]) recovered virus nucleic acids from a mixture of plant tissues (10 mg of PAV-infected A. sativa leaves and 40 mg of uninfected plant tissue from one of three host species, A. sativa , A. gerardii , or K. macrantha ) made prior to RNA extraction.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Glycoinositolphospholipids from Trypanosomatids Subvert Nitric Oxide Production in Rhodnius prolixus Salivary Glands

doi: 10.1371/journal.pone.0047285

Figure Lengend Snippet: NADPH-diaphorase activity of NOS in Rhodnius prolixus salivary glands after a blood meal and the expression of NOS. A. Salivary glands were dissected in different days after blood feeding and evaluated for NOS NAPDH-diaphorase activity. Salivary glands were assayed in 10 mM Tris-HCl pH 8,0, 0,05 M NaCl, 0,1%, Triton X-100, 1 mM CaCl 2 , 5 µM FAD, 1 mM NADPH and 0,5 mg/mL MTT. MTT reduction was followed at 540 nm for 30 min at 37°C. Also samples were obtained and NOS content evaluated by Western blotting. Each point is the average and SE of 05 different experiments. B. Immunoblotting using an anti-NOS antibody. Blottings were developed with the use of a secondary antibody conjugated to alkaline phosphatase in the presence of the substrate Western Blue. Molecular mass markers are indicated at the left. C. Upper panel , total RNA from the salivary glands at different days after feeding was isolated and cDNA was synthesized. Samples were then analyzed by semi-quantitative PCR with temperatures of 55, 72 and 94°C for 27 cycles with primers for NOS. Lower panel, analysis of 18 S RNA levels. In this case reaction occurred for 25 cycles. The products of reactions shown on panels C were separated on agarose gel 1.4% stained with ethidium bromide and photographed under ultraviolet light. Molecular mass standards are indicated at the left.

Article Snippet: Reagents Ethylenediamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), flavin -adenine dinucleotide (FAD), culture medium RPMI-1640, LIT, NADPH, Tris, glycine, acrylamide, bis-acrylamide, Tetramethylethylenediamine (TEMED), Dimethyl sulfoxide (DMSO), Dithiothreitol (DTT), bovine serum albumin, sodium SO, okadaic acid, Folin reagent and pNPP were obtained from Sigma-Aldrich Company (St. Louis, MO, USA).

Techniques: Activity Assay, Expressing, MTT Assay, Western Blot, Isolation, Synthesized, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

Journal: Molecular and Cellular Biology

Article Title: Progesterone and Glucocorticoid Receptors Recruit Distinct Coactivator Complexes and Promote Distinct Patterns of Local Chromatin Modification

doi: 10.1128/MCB.23.11.3763-3773.2003

Figure Lengend Snippet: Modulation of PR or GR activity by loss of SRC coactivator function. (A) RNA interference was carried out by introducing siRNA against individual SRCs (siSRC-1, -2, and -3) into the T47D/CAT0 cells for 3 days. Total protein was extracted with TRI reagent (Molecular Research Center, Inc). Western blotting analysis was performed to monitor the reduction of specific proteins with the indicated antibodies. The asterisk indicates a nonspecific band, used as a loading control in place of β-actin. (B) CAT activity was measured after RNA interference with the indicated siSRCs and treatment with progesterone or dexamethasone for 24 h. The uninduced or induced controls (lanes 1, 2, and 7) were treated with an unrelated siRNA against luciferase. The siSRCs labeled 1+3 and 2+3 represent the combinations of siRNA against SRC-1 and SRC-3 and SRC-2 and SRC-3, respectively. Triplicate results were normalized against results for hormone-treated controls (lanes 2 and 7), which are arbitrarily set as 100 for comparison. Analysis of variance of each individual group was done. Statistical results are mentioned in the text.

Article Snippet: Total RNA was extracted with TRI reagent (Molecular Research Center, Inc., Cincinnati, Ohio) following the manufacturer's instruction.

Techniques: Activity Assay, Western Blot, Luciferase, Labeling

Journal: Scientific Reports

Article Title: Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein

doi: 10.1038/s41598-017-04488-w

Figure Lengend Snippet: Neu3 behaves as an integral membrane protein. ( a ) Soluble (S, first lane) and membrane (P, second lane) fractions of NEU3 -transfected cells were obtained as described in Fig. 1a . The latter fraction was then incubated for 40 min on ice with Tris buffer (control) or Na 2 CO 3 at pH 11.5 in order to extract peripheral membrane proteins. After treatment, membranes were collected by ultracentrifugation, and membrane-bound (P, fourth and sixth lanes) and solubilized (S, third and fifth lanes) proteins were analyzed by Western blotting using anti-Neu3 antibody. K-Ras and Cav-1 were used as markers of an extractable and a non-extractable membrane protein, respectively. Tub was used as a marker of a soluble protein. ( b ) Intact NEU3 -transfected cells were treated with (+) or without (−) 100 µg/ml proteinase K (PK) for 30 min at 37 °C. The remaining proteins were run on a SDS-PAGE gel and Western blotted with anti-c-Myc and anti-Neu3 antibodies. Transferrin Receptor (TfR) was used as a marker of a PK-accessible protein, whereas Cav-1 and sialyltransferase ST3Gal-II were used as markers of proteins not accessible to PK. The molecular masses in kDa of the standard proteins are shown on the left. ( c ) Antibody accessibility in permeabilized and non-permeabilized cells. The signal of the indicated antibodies were analyzed after fixation in cells treated with 0.1% saponin in PBS/BSA (permeabilized) or with PBS/BSA alone (non-permeabilized). Cav-1 was used as a marker of an inner membrane protein detected only after cell permeabilization, and GPI-YFP was used as a marker of an outer membrane protein accessible by antibody without permeabilization. Cells nuclei were stained blue with Hoechst dye. ( d ) Cell surface proteins exposed to the extracellular environment of NEU3 -transfected cells were biotinylated and isolated by streptavidin agarose pull-down. Biotinylated (B) and non-biotinylated (NB) fractions were analyzed by Western blotting. Endogenous marker Cav-1 was used as control of a membrane protein not exposed to the extracellular environment. Densitometric analysis indicating the percentage of Neu3 in each fraction is shown. ( e ) Kyte-Doolittle hydrophobicity plot for Neu3 sequence using a window size of 19. The red line corresponds to a hydrophobicity score of 1.6 and designates an empirical cutoff value for α-helix transmembrane segments of ~19 amino acids long.

Article Snippet: Subcellular fractionation Cells were washed with cold PBS and harvested by scraping in 5 mM Tris/HCl pH 7.0 (buffer T) supplemented with protease inhibitor cocktail (PIC) (Sigma-Aldrich, St Louis, MO, USA).

Techniques: Transfection, Incubation, Western Blot, Marker, SDS Page, Staining, Isolation, Sequencing

Journal: mBio

Article Title: Preferential Packing of Acidic Glycosidases and Proteases into Bacteroides Outer Membrane Vesicles

doi: 10.1128/mBio.00909-14

Figure Lengend Snippet: OMV display protease activity. (A) Ten micrograms of OMV proteins was loaded on 10% Tris-glycine gel with 0.1% gelatin as the substrate. Following separation at denaturing conditions, proteins were renatured and then incubated at 37°C for 2 days to allow substrate cleavage. Colloidal Coomassie brilliant blue G-250 was used to stain the gel. Clear bands marked by the arrows indicate the digestion of gelatin by proteases. (B) Peptidase activity of B. fragilis vesicles was tested using different p -nitroanilide-linked amino acids. Ten micrograms of purified OMV proteins was incubated with different substrates for 1 h at 37°C. Activities were determined by measuring the absorbance of the released p -nitroanilide at 405 nm. All activities are represented relative to the alanine-peptidase activity. All experiments were done in triplicates.

Article Snippet: The peptidase enzyme activity was determined by incubation of 10 µg of the quantified OMV proteins in a 50 mM Tris-HCl buffer (pH 6.5), with 200 µl of 50 mM l -lysine-p -nitroanilide dihydrobromide, l -alanine-p -nitroanilide hydrochloride, l -leucine-p-nitroanilide, or pyroglutamic acid-p -nitroanilide (Sigma-Aldrich), for 1 h at 37°C.

Techniques: Activity Assay, Incubation, Staining, Purification

Journal: eLife

Article Title: The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding

doi: 10.7554/eLife.36326

Figure Lengend Snippet: SDS PAGE showing ADR1 constructs translated in PURExpress Δ-Ribosome kit supplemented with high-salt-washed ribosomes isolated from HDB140, HDB143 (uL23 Δloop), or HDB144 (uL24 Δloop) as indicated. Translations were run on 12% Bis-Tris gels with MOPS running buffer.

Article Snippet: The samples were resolved on 12% Bis-Tris gels (Thermo Scientific) in MOPS buffer for ADR1 and MES buffer for Spectrin and Titin.

Techniques: SDS Page, Construct, Isolation

Journal:

Article Title: Human Milk Adiponectin Is Associated with Infant Growth in Two Independent Cohorts

doi: 10.1089/bfm.2008.0137

Figure Lengend Snippet: Western blots of human milk adiponectin. ( A ) In native PAGE, 13 μL (13) or 10.4 μL (10) of milk (M) or serum (S) (13 μL of a 1:60 dilution) was applied to each well of a native PAGE 3–12% Bis-Tris

Article Snippet: For native polyacrylamide gele electrophoresis (PAGE), 13 μL of the aqueous milk preparation (e.g., 10.4 μL plus 2.6 μL of water) or 13 μL of a 1:60 dilution of serum was combined with 5 μL of 4× loading buffer and 2 μL of G250 and applied to each well of a native PAGE 3–12% Bis-Tris gel (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Western Blot, Clear Native PAGE

Journal: PLoS ONE

Article Title: Toxin-Antitoxin Systems in the Mobile Genome of Acidithiobacillus ferrooxidans

doi: 10.1371/journal.pone.0112226

Figure Lengend Snippet: In vitro RNase assay of ICE Afe 1 toxins. 1.6 µg of MS2 RNA was incubated with (+) or without (−) the purified toxins in 10 mM Tris-HCl (pH 7.8) in the absence of divalent ions (A) or with 10 mM MgCl 2 (B) or MnCl 2 (C). The reactions were incubated at 37°C for 15 (A and C) or 30 minutes (B). 12 mM EDTA was added to some reactions as a control (lanes 6-10).

Article Snippet: RNase activity The digestion reaction mixture (20 µl) consisted of 1.6 µg of MS2 RNA substrate (Roche) in 10 mM Tris-HCl (pH 7.8) with or without 10 mM MgCl2 or MnCl2 , 40 U RNase inhibitor Ribolock (ThermoScientific) and 100 pmol of each purified toxin.

Techniques: In Vitro, Incubation, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator

doi:

Figure Lengend Snippet: ( A ) Fibrinolytic effects of rSKs on human plasma clots. Human plasma (45 μl) was mixed with trace amounts of 125 ). After a wash with Tris-buffered saline (pH 7.4) containing the thrombin inhibitor PPACK (10 μM), the clots were suspended in 2 ml of plasma containing 10 μM PPACK. Various amounts of rSK and rSKΔ59 that had undergone active-site titration (0–50 nM) were added to the plasma, and, after 6 h, the amount of fibrinolysis was determined by measuring the release of soluble 125 ). The means ± SD are shown (but in some cases the SD are too small to be visible). ( B ) Fibrinogen degradation by rSK activator complexes. At the end of the fibrinolysis experiments presented in A ). The means ± SD are shown (but in some cases the SD are too small to be visible).

Article Snippet: After a wash with Tris-buffered saline (pH 7.4) containing the thrombin inhibitor PPACK (10 μM, d -Phe-Pro-Arg chloromethylketone, Calbiochem), the clots were suspended in 2 ml of plasma containing 10 μM PPACK.

Techniques: Titration

Journal: Arthritis and rheumatism

Article Title: Identification and Characterization of a Peptide Mimetic That May Detect a Species of Disease-Associated Anticardiolipin Antibodies in Patients With the Antiphospholipid Syndrome

doi: 10.1002/art.10836

Figure Lengend Snippet: Reactivities of CL154C with plasma samples from patients with antiphospholipid syndrome (APS) (n = 21), normal healthy donors (n = 25), and systemic lupus erythematosus (SLE) patients without APS (n = 12). Enzyme-linked immunosorbent assay plates were coated with CL154C peptide (10 μ g/ml) and plasma samples were analyzed at 1:100 dilution in Tris buffered saline/0.1% gelatin. Bound IgG was measured and reported. Each data point represents a test sample; the horizontal bars denote the mean of each group, and the dashed line represents the cutoff for positivity, that is, the mean optical density (O.D.) plus 3 SD of normal controls.

Article Snippet: After washing with Tris buffered saline (TBS), bound phage in the wells were eluted with 35 μ l elution buffer (0.1 M HCl adjusted to pH 2.2 with glycine, and containing 1 mg/ml bovine serum albumin [BSA]; fraction V; Sigma, St. Louis, MO) per well, followed by incubation for 10 minutes at room temperature (RT).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Nucleic Acids Research

Article Title: Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding

doi: 10.1093/nar/gkh743

Figure Lengend Snippet: HCV helicase catalyzed DNA unwinding in various buffer systems. ( A ) 1 nM of a DNA substrate was incubated with 100 nM of HCV helicase (His–Hel) and 5 mM ATP for 10 min at 23°C in either citrate buffer (squares), MES-NaOH buffer (triangles), PIPES-NaOH buffer (diamonds), MOPS-NaOH buffer (circles), Tris–HCl buffer (×), Tris–Malate buffer (+) or Tricene-HCl ( * ). Data are reported as percent of substrate unwound. ( B ) Native polyacrylamide gels showing the time course of the reactions catalyzed in MOPS buffers at pH 6.25, 7.04 and 7.68. Reactions were terminated at various times are shown along with a boiled substrate control.

Article Snippet: 3-(Cyclohexylamino)propane sulfonic acid (CAPS), N -Tris(hydroxymethyl)methylglycine (TRICINE), Tris, MOPS, PIPES and morpholinoethane sulfonic acid (MES) buffers were each prepared at the same ionic strength and treated with RNAsecure reagent (Ambion) prior to use.

Techniques: Incubation

Journal: Nucleic Acids Research

Article Title: Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding

doi: 10.1093/nar/gkh743

Figure Lengend Snippet: Effect of pH on HCV helicase catalyzed ATP hydrolysis. ( A ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the absence of nucleic acid as a function of pH. ( B ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the presence of saturating 2 mM poly(U) RNA. In both (A) and (B), reactions were performed in MES buffer (squares), PIPES buffer (triangles), MOPS buffer (diamonds), TRIS buffer (circles), Tricene buffer (×) or CAPS buffer (+). Average rate constants from four separate determinations are shown with the standard deviations as error bars.

Article Snippet: 3-(Cyclohexylamino)propane sulfonic acid (CAPS), N -Tris(hydroxymethyl)methylglycine (TRICINE), Tris, MOPS, PIPES and morpholinoethane sulfonic acid (MES) buffers were each prepared at the same ionic strength and treated with RNAsecure reagent (Ambion) prior to use.

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: Heterologous Production of Methionine-?-Lyase from Brevibacterium linens in Lactococcus lactis and Formation of Volatile Sulfur Compounds ▿ and Formation of Volatile Sulfur Compounds ▿ †

doi: 10.1128/AEM.02417-08

Figure Lengend Snippet: Cysteine desulfhydrase activity by in situ staining. MGL activity was monitored in Tris-glycine gels under nondenaturing conditions using l -cysteine as the substrate. Lanes: M, molecular mass markers; 1, purified recombinant His-tagged MGL protein; 2,

Article Snippet: The activities of purified His-tagged MGL or CFEs from recombinant L. lactis cultures were monitored using Tris-glycine gels (Invitrogen) run under nondenaturing conditions.

Techniques: Activity Assay, In Situ, Staining, Purification, Recombinant

Journal: Nucleic Acids Research

Article Title: Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase

doi: 10.1093/nar/gkw656

Figure Lengend Snippet: RNAP containing βG1249D generates holoenzyme with AsiA/σD581BpA, but is defective in generating the crosslink with β’. ( A ) Native Tris-glycine gel. Solutions were assembled with the indicated components. The positions of AsiA, RNAP core, AsiA-RNAP holoenzyme, and σD581BpA are marked. (The bands that migrate faster than AsiA seen in lane 1 are trace contaminants present in the σD581BpA preparation.) ( B ) SDS-PAGE gel showing the products obtained after photocrosslinking. Arrow points to the crosslink between σD581BpA and β’ 80 HRGVICEK 87 identified in Figure 3 .

Article Snippet: A 20 μl aliquot was used for photocrosslinking, and a 5 μl aliquot was applied to a 4–12% Tris-glycine gel (Invitrogen/Thermo Fisher) run in 1× Native Tris-glycine buffer (Invitrogen/Thermo Fisher) and stained in Gel Code (Thermo Fisher) as described ( ).

Techniques: SDS Page

Journal: PLoS Genetics

Article Title: Transcriptional Control in Cardiac Progenitors: Tbx1 Interacts with the BAF Chromatin Remodeling Complex and Regulates Wnt5a

doi: 10.1371/journal.pgen.1002571

Figure Lengend Snippet: Effects of Tbx1 on H3K4 di- and tri-methylation and H3 acethylation status of the TBE regions of Wnt5a . q-ChIP assay from P19Cl6 cells transfected with an empty vector (EV) or with a vector over-expressing Tbx1 (Tbx1) followed by quantitative real-time PCR. The immunoprecipitation was carried out with anti-H3K4me2, anti-H3K4me3 or anti-AcH3 antibodies. (A) TBE1/2 region, (B) TBE3 region, and (C) p16 promoter region (positive control). There is no enrichment for any of these histone modifications at the TBE regions. Values are from 3 experiments (mean±S.D.).

Article Snippet: RNA extraction, cDNA synthesis, and Q-RT-PCR RNA was extracted from P19Cl6 cells using TRI-Reagent (Ambion/Applied Biosystems) according to the manufacturer's protocol.

Techniques: Methylation, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, Positive Control

Journal: Scientific Reports

Article Title: The role of irradiance and C-use strategies in tropical macroalgae photosynthetic response to ocean acidification

doi: 10.1038/s41598-018-27333-0

Figure Lengend Snippet: Inhibition of P gmax in three fleshy ( a ) and five calcifying ( b ) tropical macroalgae as inhibitor block various bicarbonate uptake pathways: external carbonic anhydrase (CA ext ) with acetazolamide (AZ), AE protein by pyridoxal (5) phosphate (PLP), proton pump acidification by Tris buffer and ATPase H + pumps by sodium orthovanadate.

Article Snippet: Inhibitors included acetazolamide (AZ, Sigma Aldrich) that blocks the dehydration of HCO3 − into CO2 via external carbonate anhydrase (CAext ) , pyridoxal (5) phosphate (PLP, Fisher Scientific) that inhibits active uptake of HCO3 − , Tris buffer (Trizma R, Sigma Aldrich) that interferes with proton pump acidification of the thalli boundary layer and sodium orthovanadate (vanadate, Sigma Aldrich) that obstructs plasmalemma ATPase H+ pumps .

Techniques: Inhibition, Blocking Assay, Plasmid Purification

Journal: Scientific Reports

Article Title: Polysulfide Na2S4 regulates the activation of PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4-naphthoquinone through formation of sulfur adducts

doi: 10.1038/s41598-017-04590-z

Figure Lengend Snippet: Chemical modification of cellular and recombinant PTEN by 1,4-NQ and suppression of 1,4-NQ modification of cellular PTEN. ( A ) Primary mouse hepatocytes were simultaneously treated with DMSO or 1,4-NQ and 10 (upper) or 100 (lower) µM Na 2 S 4 for 30 min, then PTEN was immunoprecipitated using anti-PTEN antibody. Western blotting was performed using the indicated antibodies. Representative blots are shown from three independent experiments. ( B ) Recombinant GST-tagged human PTEN (1 μg) was incubated with 1,4-NQ (1–8 μM) at 25 °C for 1 h. The reaction mixture was then subjected to immunoblotting, with the anti-1,4-NQ antibody, and SDS-PAGE, with Coomassie Brilliant Blue staining. Representative blots are shown from three independent experiments. ( C ) Results of nanoUPLC-MS E analysis of 1,4-NQ-modified cysteine residues in GST-tagged human PTEN. Recombinant GST-tagged human PTEN (1.7 μg) was incubated with 1,4-NQ (10 μM) at 25 °C for 30 min in a total volume of 10 μL 50 mM Tris-HCl (pH 7.5). After the reaction, PTEN protein was digested with trypsin and analyzed by nanoUPLC-MS E . The corresponding MS E data are shown in Table 1 .

Article Snippet: Anti-rabbit IgG conjugated magnetic beads (100 µL, Dynabeads M-280-sheep anti-rabbit IgG) were washed three times with Tris-buffered saline and Tween 20, then incubated with anti-PTEN antibodies (5 µL, Cell Signaling Technology, #9552) at 4 °C for 3 h. The unbound antibodies were removed and the beads resuspended in 500 µL cell lysate (1 µg/µL).

Techniques: Modification, Recombinant, Immunoprecipitation, Western Blot, Incubation, SDS Page, Staining, Mass Spectrometry

Journal: PLoS ONE

Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

doi: 10.1371/journal.pone.0060245

Figure Lengend Snippet: Humoral immune responses in p24CE DNA vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).

Article Snippet: The proteins were resolved on 10% or 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA), transferred onto nitrocellulose membranes (Invitrogen), which were probed with a goat anti-p24gag antibody (dilution 1∶3000, provided by L. Arthur, SAIC, NCI, Frederick) followed by anti-goat IgG-HRP labeled antibody (dilution 1∶10,000; Calbiochem, EMD chemicals, Gibbstown, NJ) or with plasma (1∶200 dilution) from DNA vaccinated mice followed by anti-mouse IgG-HRP labeled (1∶10,000 dilution, GE Healthcare, Piscataway, NJ).

Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Labeling, Expressing

Journal: PLoS ONE

Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

doi: 10.1371/journal.pone.0060245

Figure Lengend Snippet: Expression of the p24CE plasmids upon transient transfection in cultured cells. Plasmid DNA (1 µg) expressing different variants of either p24CE1 (left panel) or p24CE2 (right panel) proteins were transfected in HEK293 cells. The cultures were harvested 24 hrs later and proteins from equal amounts (1/250) from the cell-associated (top panel) and extra-cellular (bottom panel) fractions were resolved on a 12% NuPAGE Bis-Tris gel and analyzed by Western immunoblot using a goat anti-p24 gag antiserum and visualized using enhanced ECL. The membrane containing the cell-associated fractions was also probed with anti-human pan actin antibody to control for equal loading of the samples.

Article Snippet: The proteins were resolved on 10% or 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA), transferred onto nitrocellulose membranes (Invitrogen), which were probed with a goat anti-p24gag antibody (dilution 1∶3000, provided by L. Arthur, SAIC, NCI, Frederick) followed by anti-goat IgG-HRP labeled antibody (dilution 1∶10,000; Calbiochem, EMD chemicals, Gibbstown, NJ) or with plasma (1∶200 dilution) from DNA vaccinated mice followed by anti-mouse IgG-HRP labeled (1∶10,000 dilution, GE Healthcare, Piscataway, NJ).

Techniques: Expressing, Transfection, Cell Culture, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Amyloid-? Peptide Binds to Cytochrome C Oxidase Subunit 1

doi: 10.1371/journal.pone.0042344

Figure Lengend Snippet: Western blot analysis of recombinant C2 phage expressing the fragment of CcOX1. 10 11 phage particles diluted in loading buffer were resolved on 4–12% NuPAGE Bis-Tris gel (Invitrogen) at 200 V for 45 min at room temperature and immunoblotted for detection with anti-pIII antibody. Wild-type M13 phage was used as a control. Migration of the molecular mass standards as well as pIII and pIII–CcOX1 fusion protein are indicated by arrowheads.

Article Snippet: 1011 phage particles diluted in 16 µl of loading buffer were boiled 5 minutes and separated on 4–12% NuPAGE Bis-Tris gel (Invitrogen) at 200 V for 45 min at room temperature as recommended by manufacturer.

Techniques: Western Blot, Recombinant, Expressing, Migration

Journal: PLoS Pathogens

Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

doi: 10.1371/journal.ppat.1007006

Figure Lengend Snippet: Biochemical characterization of the recombinant protein LuloHya. (A) Purification of LuloHya by size exclusion chromatography using Superdex 200 Increase 10/300 GL column. (B) Coomassie-stained gel electrophoresis of LuloHya (1 μg). Mouse anti-LuloHya antibodies (1:5,000) recognized LuloHya (100 ng) and a single band in the SGE (5 pairs of SG) by Western blot. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) NuPAGE Novex 4–12% Bis-Tris protein gel shows differences in electrophoretic pattern of LuloHya (1 μg) and its deglycosylated form (deLuloHya: 1 μg) which runs at the expected molecular weight (42.3 kDa). M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (D) Hyaluronidase activity of 10 nM LuloHya and its deglycosylated form (deLuloHya). As negative controls, 4 micrograms of HA were incubated with TBS instead of recombinant protein or the deglycosylation enzyme mix (DeglycoMx Kit, QABio) without protein. (E) Turbidimetric assay showed a clear pH dependence of hyaluronidase activity of LuloHya. Reaction mixtures were prepared with solutions containing 25 mM buffer (described in Methods), 100 mM NaCl, 0.1% BSA, and different pH values (4–12.5; adjusted with a pHmeter 430, Corning). (F) Ionic strength dependency was analyzed in reaction mixtures of 25 mM HEPES, 0.1% BSA, pH 7.3 with variable NaCl concentration (25–1000 mM). Hyaluronidase activity is inversely expressed as the remaining HA (%) after treatment of 4 μg of HA with 10 nM enzyme during 1 h at 37°C. Biological triplicates and technical duplicates were assessed. Multiple comparisons were done by one-way ANOVA (****: p

Article Snippet: Proteins were transferred to a nitrocellulose membrane (iBlot, Invitrogene) that was blocked overnight at 4°C with blocking buffer: 50 mM Tris, 150 mM NaCl containing 5% (w/v) powdered nonfat blotting-grade milk (Bio-Rad) and 0.05% of Tween-20 (Sigma).

Techniques: Recombinant, Purification, Size-exclusion Chromatography, Staining, Nucleic Acid Electrophoresis, Western Blot, Molecular Weight, Activity Assay, Incubation, Turbidimetric Assay, Concentration Assay

Journal: Journal of bioenergetics and biomembranes

Article Title: Minocycline chelates Ca2+, binds to membranes, and depolarizes mitochondria by formation of Ca2+-dependent ion channels

doi: 10.1007/s10863-010-9271-1

Figure Lengend Snippet: (Panel A ) Minocycline (20 µM) in the presence of 3 mM Ca 2+ induces an electrical current in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 100 mM KCl, pH 8.5; the voltage was 30 mV. (Panels B, C ) Ion channel activity induced by minocycline (8 µM) in the presence of Ca 2+ (3 mM in panel B and 20 µM in panel C) at 50 mV in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 1 M KCl, pH 9.0. (A current of 0.7 pA at 50 mV corresponds to a conductance of 14 pS)

Article Snippet: β-Alanine, micro Biuret protein assay kit, bovine serum albumin (fatty acid free grade Sigma A-6003), CaCl2 , EGTA, gramicidin A, Hepes (ultra grade), minocycline, KCl, KOH, KH2 PO4 , MES, Ruthenium 360 (Ru360), succinate, sucrose (ultra grade), PBS-Tween buffer, and Tris-base were obtained from Sigma (St. Louis, MO, USA).

Techniques: Activity Assay

Journal: Nature Communications

Article Title: A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers

doi: 10.1038/s41467-018-03509-0

Figure Lengend Snippet: The A11(+) oligomers bind to the 20S proteasome and impair opening of the substrate gate. a 20S proteasomes (0.4 μg) and pure non-crosslinked Aβ*56 oligomers (1.5 μg) were incubated separately or together for 30 min (37 °C), crosslinked with 1 mM glutaraldehyde for 5 min, and separated by Native-PAGE (4–8% Tris–acetate gel). Total protein was detected by silver stain (left), and total Aβ was detected by western blot (right). b – d The activity of yeast 20S wild-type (WT) and open-gate (α3ΔN) proteasomes was measured for all three proteolytic sites in the presence of A11(+) oligomers from Aβ*56 ( b ; 2.5 μM), α-Syn ( c ; 100 nM), and Htt-53Q ( d ; 50 nM). Chymotrypsin-like activity was measured by LLVY-amc hydrolysis, trypsin-like activity by RLR-amc, and caspase like by nLPnLD-amc hydrolysis. The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass. All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation

Article Snippet: SDS-PAGE and Native-PAGE Proteins were separated by SDS-PAGE using NuPAGE™ 4–12% Bis-Tris protein gels (Invitrogen), or separated by Native-PAGE using Novex™ 10–20% Tris-glycine or NuPAGE™ 3–8% Tris–acetate protein gels (Invitrogen), as indicated.

Techniques: Incubation, Clear Native PAGE, Silver Staining, Western Blot, Activity Assay, Standard Deviation