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  • 99
    Thermo Fisher sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to <t>SDS-PAGE,</t> and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6011 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by <t>SDS-PAGE</t> and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21562 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    Protein analysis of M. tuberculosis. (A) <t>SDS-PAGE</t> analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 12745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore immobilon p pvdf membranes
    Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to <t>PVDF</t> membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.
    Immobilon P Pvdf Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher bis tris sds page gels
    New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates <t>(SDS</t> fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing <t>SDS/PAGE</t> and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.
    Bis Tris Sds Page Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher bis tris sds page gel
    Immunodetection of Cr LPAAT 1 expression in C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The <t>SDS</t> ‐ <t>PAGE</t> gel was stained with blue dye (ProSieve EX Safe Stain— LONZA ) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS ‐ PAGE gel, and expression of Cr LPAAT 1 was detected using anti‐ HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE : pLM 21‐Cr LPAAT 1‐ HA overexpressors; three transformants ( OE 1, OE 2 and OE 3) are shown.
    Bis Tris Sds Page Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1180 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sodium dodecyl sulfate solution
    Expression profiles of four DNA polymerases in Sulfolobus islandicus . Exponential growth cultures (A600 = 0.2) of the wild-type (WT) strain, S. islandicus E233S was treated with DNA damage agent (2 μM of NQO) or untreated (CK), and these cultures were then grown for 24 h during which cell samples were taken at indicated time points (hours) and used for preparation of cell extracts. Proteins in the cell extracts (10 μg) were fractionated by <t>sodium</t> <t>dodecyl</t> <t>sulfate–polyacrylamide</t> gel electrophoresis (SDS-PAGE), and DNA polymerases were identified by western blotting and hybridization using antibodies raised against each DNA polymerase. PCNA1, a subunit of the archaeal replication sliding clamp, serving as a loading control.
    Sodium Dodecyl Sulfate Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P

    Journal: Infection and Immunity

    Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

    doi: 10.1128/IAI.00004-09

    Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P

    Article Snippet: Samples were separated by 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Nupage Bis-Tris gel; Invitrogen) and electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany).

    Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

    Analysis of LPS-Trap-Fc1 to -Fc4. (A) Schematic representation of the mTLR4/mMD-2 fusion proteins LPS-Trap (left top) and LPS-Trap-Fc1 (left bottom and right). (B) Supernatants of LPS-Trap-Fc1-, -Fc2-, -Fc3-, or -Fc4-transfected HEK293T cells were subjected to SDS-PAGE under reducing conditions and analyzed by Western blotting for the human IgG-Fc tail (left) or the N-terminal FLAG epitope (right).

    Journal: Infection and Immunity

    Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

    doi: 10.1128/IAI.00004-09

    Figure Lengend Snippet: Analysis of LPS-Trap-Fc1 to -Fc4. (A) Schematic representation of the mTLR4/mMD-2 fusion proteins LPS-Trap (left top) and LPS-Trap-Fc1 (left bottom and right). (B) Supernatants of LPS-Trap-Fc1-, -Fc2-, -Fc3-, or -Fc4-transfected HEK293T cells were subjected to SDS-PAGE under reducing conditions and analyzed by Western blotting for the human IgG-Fc tail (left) or the N-terminal FLAG epitope (right).

    Article Snippet: Samples were separated by 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Nupage Bis-Tris gel; Invitrogen) and electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany).

    Techniques: Transfection, SDS Page, Western Blot, FLAG-tag

    Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by SDS-PAGE and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.

    Journal: Gene

    Article Title: Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus

    doi: 10.1016/j.gene.2017.06.048

    Figure Lengend Snippet: Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by SDS-PAGE and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.

    Article Snippet: The proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting ( B), using a primary antibody of rabbit anti-6 × His (Sigma) and a secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma).

    Techniques: Expressing, Transfection, Purification, Column Chromatography, SDS Page, Western Blot, Marker

    Construction of pET-28a-S7 vector and identification of recombinant VP7 and prepared polyclonal VP7 antibody. (A) Electrophoretogram. (B) SDS-PAGE and Western blotting.

    Journal: Gene

    Article Title: Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus

    doi: 10.1016/j.gene.2017.06.048

    Figure Lengend Snippet: Construction of pET-28a-S7 vector and identification of recombinant VP7 and prepared polyclonal VP7 antibody. (A) Electrophoretogram. (B) SDS-PAGE and Western blotting.

    Article Snippet: The proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting ( B), using a primary antibody of rabbit anti-6 × His (Sigma) and a secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma).

    Techniques: Positron Emission Tomography, Plasmid Preparation, Recombinant, SDS Page, Western Blot

    Transforming activity of a PH domain-minus derivative of Dbs is restored by the addition of a plasma membrane-targeting signal from H-Ras. (A) The domain structure of the full-length Dbs protein is illustrated in the upper line (DH, Dbl homology domain; PH, pleckstrin homology domain; SH3, Src homology 3 domain), and the lines below indicate the regions of the protein included in predicted translation products of the cDNA derivatives. The numbers refer to amino acid positions in the full-length protein. The Dbs-HA7 derivative is fused to the H-Ras COOH-terminal plasma membrane-targeting sequence that includes the CAAX tetrapeptide isoprenylation signal (designated CAAX). All derivatives were fused at their NH 2 termini to an HA epitope tag. The values at the right indicate the transforming activity of the different derivatives in NIH 3T3 cell focus formation assays. The data shown are representative of three independent assays done on triplicate plates. (B) Expression of HA epitope-tagged Dbs proteins. Epitope-tagged proteins were expressed transiently in 293 cells (left-hand lanes) or stably in NIH 3T3 cells (right-hand lanes) and detected by Western blotting with an anti-HA epitope antibody. (C) Subcellular localization of PH domain variants of Dbs. HA epitope-tagged proteins were expressed stably in NIH 3T3 cells. The cells were lysed and fractionated at 100,000 × g . Thirty micrograms of protein from total (T), soluble (S), and particulate (P) fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein expression was determined with the anti-HA epitope antibody (BabCo). The relative amounts of protein in particulate and soluble fractions (%) were determined with a PhosphorImager (Molecular Dynamics).

    Journal: Molecular and Cellular Biology

    Article Title: Dependence of Dbl and Dbs Transformation on MEK and NF-?B Activation

    doi:

    Figure Lengend Snippet: Transforming activity of a PH domain-minus derivative of Dbs is restored by the addition of a plasma membrane-targeting signal from H-Ras. (A) The domain structure of the full-length Dbs protein is illustrated in the upper line (DH, Dbl homology domain; PH, pleckstrin homology domain; SH3, Src homology 3 domain), and the lines below indicate the regions of the protein included in predicted translation products of the cDNA derivatives. The numbers refer to amino acid positions in the full-length protein. The Dbs-HA7 derivative is fused to the H-Ras COOH-terminal plasma membrane-targeting sequence that includes the CAAX tetrapeptide isoprenylation signal (designated CAAX). All derivatives were fused at their NH 2 termini to an HA epitope tag. The values at the right indicate the transforming activity of the different derivatives in NIH 3T3 cell focus formation assays. The data shown are representative of three independent assays done on triplicate plates. (B) Expression of HA epitope-tagged Dbs proteins. Epitope-tagged proteins were expressed transiently in 293 cells (left-hand lanes) or stably in NIH 3T3 cells (right-hand lanes) and detected by Western blotting with an anti-HA epitope antibody. (C) Subcellular localization of PH domain variants of Dbs. HA epitope-tagged proteins were expressed stably in NIH 3T3 cells. The cells were lysed and fractionated at 100,000 × g . Thirty micrograms of protein from total (T), soluble (S), and particulate (P) fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein expression was determined with the anti-HA epitope antibody (BabCo). The relative amounts of protein in particulate and soluble fractions (%) were determined with a PhosphorImager (Molecular Dynamics).

    Article Snippet: The protein concentrations of the total, cytosolic, and membrane fractions were determined with a biciuchoninic acid protein assay kit (Pierce) with 30 μg of protein for each fraction, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore), and probed with anti-HA epitope antibody (BabCo).

    Techniques: Activity Assay, Sequencing, Expressing, Stable Transfection, Western Blot, Polyacrylamide Gel Electrophoresis

    CD spectra of Cpl ( A ) and melittin ( B ). The peptides were dissolved in 10 mM PBS (pH 7.4), 50% TFE, and 30 mM SDS. Data are expressed as the mean residue ellipticities.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Broad-Spectrum Antimicrobial Activity and Low Cytotoxicity against Human Cells of a Peptide Derived from Bovine αS1-Casein

    doi: 10.3390/molecules23051220

    Figure Lengend Snippet: CD spectra of Cpl ( A ) and melittin ( B ). The peptides were dissolved in 10 mM PBS (pH 7.4), 50% TFE, and 30 mM SDS. Data are expressed as the mean residue ellipticities.

    Article Snippet: Sodium dodecyl sulfate (SDS), trifluoroethyl alcohol (TFE), Triton X-100, o-nitrophenyl-b-d-galactopyranoside (ONPG), 3,30-dipropylthiadicarbocyanine (diSC3-5), HEPES, N -phenyl-1-napthylamine (NPN), propidium iodide (PI) were obtained from Sigma-Aldrich (Shanghai, China).

    Techniques:

    Sodium dodecyl sulfate-polyacrylamide gels (A) and Western immunoblots (B to D) of different hantavirus N proteins with HTNV-positive human sera. (B) Detection of IgG antibodies; (C and D) detection of IgM antibodies. The lanes contain recombinant yeast-expressed

    Journal:

    Article Title: Use of Saccharomyces cerevisiae-Expressed Recombinant Nucleocapsid Protein To Detect Hantaan Virus-Specific Immunoglobulin G (IgG) and IgM in Oral Fluid ▿

    doi: 10.1128/CVI.00188-07

    Figure Lengend Snippet: Sodium dodecyl sulfate-polyacrylamide gels (A) and Western immunoblots (B to D) of different hantavirus N proteins with HTNV-positive human sera. (B) Detection of IgG antibodies; (C and D) detection of IgM antibodies. The lanes contain recombinant yeast-expressed

    Article Snippet: Protein samples were separated in 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA) by semidry blotting.

    Techniques: Western Blot, Recombinant

    Production of Nb displaying LVs. ( a ) Non-modified HEK 293T cells or HEK 293T cells stably expressing Nb BCII10 or DC2.1 were used to produce LVs pseudotyped with VSV.G or VSV.GS, respectively. Three days after transfection of these cells with the VSV.G or VSV.GS, gag/pol and transgene encoding plasmids, we evaluated the expression of the transgene (Thy1.1 or tNGFR), as well as the Nb (myc tag) by flow cytometry. Non-transfected HEK 293T cells served as a control. The flow cytometry dot plots demonstrate high expression of the transgene ( y axis) in all transfected cells (VSV.G, BCII10 and DC2.1) and high expression of Nbs ( x -axis) on the Nb-modified HEK 293T cells (BCII10 and DC2.1). One representative experiment is shown ( n =6). ( b ) To compare the LV preparations we determined their RT content. The graph depicts the amount of RT (ng RT/μl) in the LV preparations. Each dot represents one LV stock, the horizontal line shows the mean ( n =6). ( c ) An ELISA involving anti-VHH and anti-Thy1.1 as capture and detection antibodies, respectively, was used to demonstrate the incorporation of Nbs into the surface of Thy1.1 encoding LVs. A serial dilution of LVs was applied (5, 2.5 and 1.25 ng RT). The graph depicts the OD-values detected. One representative experiment is shown ( n =3). ( d ) Western blot was performed as a quality control of the LVs. After separation on a 15% sodium dodecyl sulphate-polyacrylamide gel and transfer to a nitrocellulose membrane, the Nbs (±25 kDa) and VSV.GS (±15 kDa), which both contain an HA epitope tag, were detected with an anti-HA antibody. One representative experiment is shown ( n =4). ( e ) The density of the western blot signals was determined using the Photocapt MW software and used to determine the ratio of Nbs/VSV.GS on the LVs. This ratio is shown in the graph, in which each dot represents one LV stock and the horizontal line shows the mean ( n =4).

    Journal: Gene Therapy

    Article Title: Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells

    doi: 10.1038/gt.2011.206

    Figure Lengend Snippet: Production of Nb displaying LVs. ( a ) Non-modified HEK 293T cells or HEK 293T cells stably expressing Nb BCII10 or DC2.1 were used to produce LVs pseudotyped with VSV.G or VSV.GS, respectively. Three days after transfection of these cells with the VSV.G or VSV.GS, gag/pol and transgene encoding plasmids, we evaluated the expression of the transgene (Thy1.1 or tNGFR), as well as the Nb (myc tag) by flow cytometry. Non-transfected HEK 293T cells served as a control. The flow cytometry dot plots demonstrate high expression of the transgene ( y axis) in all transfected cells (VSV.G, BCII10 and DC2.1) and high expression of Nbs ( x -axis) on the Nb-modified HEK 293T cells (BCII10 and DC2.1). One representative experiment is shown ( n =6). ( b ) To compare the LV preparations we determined their RT content. The graph depicts the amount of RT (ng RT/μl) in the LV preparations. Each dot represents one LV stock, the horizontal line shows the mean ( n =6). ( c ) An ELISA involving anti-VHH and anti-Thy1.1 as capture and detection antibodies, respectively, was used to demonstrate the incorporation of Nbs into the surface of Thy1.1 encoding LVs. A serial dilution of LVs was applied (5, 2.5 and 1.25 ng RT). The graph depicts the OD-values detected. One representative experiment is shown ( n =3). ( d ) Western blot was performed as a quality control of the LVs. After separation on a 15% sodium dodecyl sulphate-polyacrylamide gel and transfer to a nitrocellulose membrane, the Nbs (±25 kDa) and VSV.GS (±15 kDa), which both contain an HA epitope tag, were detected with an anti-HA antibody. One representative experiment is shown ( n =4). ( e ) The density of the western blot signals was determined using the Photocapt MW software and used to determine the ratio of Nbs/VSV.GS on the LVs. This ratio is shown in the graph, in which each dot represents one LV stock and the horizontal line shows the mean ( n =4).

    Article Snippet: Viral proteins were separated on a 15% sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane, after which Nbs and VSV.GS, which contain an HA tag, were detected with an anti-HA antibody (Sigma-Aldrich) and a horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology, Heidelberg, Germany) as primary and secondary antibody, respectively.

    Techniques: Modification, Stable Transfection, Expressing, Transfection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Serial Dilution, Western Blot, Software

    Biochemical and morphological properties of rPrP-res RNA and rPrP-res RNA-low . ( A ) Two hundred microliters of sPMCA products of rPrP-res RNA (R) and rPrP-res RNA-low (R-low) were centrifuged at 100,000 x g at 4°C for 1 h. The supernatant was collected and the pellet was resuspended in 200 μL of PMCA buffer. For the total (T), supernatant (S), and pellet (P) fractions, 1 μL of each fraction was loaded onto SDS-PAGE; for PK digested samples, 10 μL of S or P was subjected to PK digestion followed by SDS-PAGE and western blotting. ( B ) Representative atomic force microscopy images of different rPrP-res aggregates. Control sample contains cofactors only in the absence of rPrP. The sample was processed in identical way as those containing rPrP. Upper panels are images of Control, rPrP-res RNA-low (R-low), rPrP-res RNA (R) and sonicated rPrP-res RNA (R (Sonicated)) at lower magnification. Bottom panels are images of rPrP-res RNA-low (R-low), rPrP-res RNA (R) and sonicated rPrP-res RNA (R (Sonicated)) at higher magnification. Bars at each panel represent 400 nm.

    Journal: PLoS Pathogens

    Article Title: Self-propagating, protease-resistant, recombinant prion protein conformers with or without in vivo pathogenicity

    doi: 10.1371/journal.ppat.1006491

    Figure Lengend Snippet: Biochemical and morphological properties of rPrP-res RNA and rPrP-res RNA-low . ( A ) Two hundred microliters of sPMCA products of rPrP-res RNA (R) and rPrP-res RNA-low (R-low) were centrifuged at 100,000 x g at 4°C for 1 h. The supernatant was collected and the pellet was resuspended in 200 μL of PMCA buffer. For the total (T), supernatant (S), and pellet (P) fractions, 1 μL of each fraction was loaded onto SDS-PAGE; for PK digested samples, 10 μL of S or P was subjected to PK digestion followed by SDS-PAGE and western blotting. ( B ) Representative atomic force microscopy images of different rPrP-res aggregates. Control sample contains cofactors only in the absence of rPrP. The sample was processed in identical way as those containing rPrP. Upper panels are images of Control, rPrP-res RNA-low (R-low), rPrP-res RNA (R) and sonicated rPrP-res RNA (R (Sonicated)) at lower magnification. Bottom panels are images of rPrP-res RNA-low (R-low), rPrP-res RNA (R) and sonicated rPrP-res RNA (R (Sonicated)) at higher magnification. Bars at each panel represent 400 nm.

    Article Snippet: For RT-QuIC analysis, BHs or PMCA products were serially diluted in 0.1% SDS (sodium dodecyl sulfate, Sigma)/N2 (Gibco)/PBS as previously reported[ ] with the last dilutions prepared to a final concentration of 0.05% SDS/N2/PBS.

    Techniques: SDS Page, Western Blot, Microscopy, Sonication

    The propagation of both pathogenic rPrP-res RNA and non-pathogenic rPrP-res RNA-low is cofactor-dependent. ( A ) In seeded sPMCA reactions, rPrP-res RNA (R) or rPrP-res RNA-low (R-low) were added to complete substrates (+cofactors) and to substrates without any cofactor (-cofactors). The mixtures were subjected to 3 rounds of sPMCA as indicated. After each round, 10 μL of PMCA product was collected and subjected to PK digestion, SDS-PAGE, and western blotting. C: undigested rPrP as controls. ( B ) Six rounds of sPMCA seeded by rPrP-res RNA with (+cofactors) or without cofactors (-cofactors). After each round, 10 μL of PMCA product was collected and subjected to PK digestion, SDS-PAGE, and western blotting. C: rPrP as controls. ( C ) The Elispot cell infection assay of round-6 sPMCA products from panel B .

    Journal: PLoS Pathogens

    Article Title: Self-propagating, protease-resistant, recombinant prion protein conformers with or without in vivo pathogenicity

    doi: 10.1371/journal.ppat.1006491

    Figure Lengend Snippet: The propagation of both pathogenic rPrP-res RNA and non-pathogenic rPrP-res RNA-low is cofactor-dependent. ( A ) In seeded sPMCA reactions, rPrP-res RNA (R) or rPrP-res RNA-low (R-low) were added to complete substrates (+cofactors) and to substrates without any cofactor (-cofactors). The mixtures were subjected to 3 rounds of sPMCA as indicated. After each round, 10 μL of PMCA product was collected and subjected to PK digestion, SDS-PAGE, and western blotting. C: undigested rPrP as controls. ( B ) Six rounds of sPMCA seeded by rPrP-res RNA with (+cofactors) or without cofactors (-cofactors). After each round, 10 μL of PMCA product was collected and subjected to PK digestion, SDS-PAGE, and western blotting. C: rPrP as controls. ( C ) The Elispot cell infection assay of round-6 sPMCA products from panel B .

    Article Snippet: For RT-QuIC analysis, BHs or PMCA products were serially diluted in 0.1% SDS (sodium dodecyl sulfate, Sigma)/N2 (Gibco)/PBS as previously reported[ ] with the last dilutions prepared to a final concentration of 0.05% SDS/N2/PBS.

    Techniques: SDS Page, Western Blot, Enzyme-linked Immunospot, Infection

    (A) dsRNA-1 and dsRNA-2 target sites within golgin-97 mRNA. (B) RNAi dose response curve. Hela cells were transfected with indicated amounts of dsRNA-1, dsRNA-2, or the negative RNAi control. The relative levels of golgin-97 expression, “dsRNA-1” or “dsRNA-2” versus “Negative RNAi control” were evaluated by immunoblot analysis. (C) Immunoblot analysis of golgin-97 expression in the presence of golgin-97 targeting dsRNAs. Normalized samples transfected with 30 pmol of dsRNA-1 and the negative RNAi control (“NC”) or 20 pmol of dsRNA-2 and the negative RNAi control (“NC”) were separated on SDS-PAGE, blotted, and incubated either with anti-golgin-97 antibodies or anti-actin antibodies used as a control for non-specific RNAi effect on expression of unrelated proteins.

    Journal: Virology

    Article Title: A host cell membrane protein, golgin-97, is essential for poxvirus morphogenesis

    doi: 10.1016/j.virol.2007.01.003

    Figure Lengend Snippet: (A) dsRNA-1 and dsRNA-2 target sites within golgin-97 mRNA. (B) RNAi dose response curve. Hela cells were transfected with indicated amounts of dsRNA-1, dsRNA-2, or the negative RNAi control. The relative levels of golgin-97 expression, “dsRNA-1” or “dsRNA-2” versus “Negative RNAi control” were evaluated by immunoblot analysis. (C) Immunoblot analysis of golgin-97 expression in the presence of golgin-97 targeting dsRNAs. Normalized samples transfected with 30 pmol of dsRNA-1 and the negative RNAi control (“NC”) or 20 pmol of dsRNA-2 and the negative RNAi control (“NC”) were separated on SDS-PAGE, blotted, and incubated either with anti-golgin-97 antibodies or anti-actin antibodies used as a control for non-specific RNAi effect on expression of unrelated proteins.

    Article Snippet: Proteins were separated by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF-membrane (Immobilon-PSQ , Millipore, Billerica, MA).

    Techniques: Transfection, Expressing, SDS Page, Incubation

    A: PPARγ activators decrease MMP-9 gelatinolytic activity in supernatants from monocyte-derived macrophages (MØ) in a concentration-dependent fashion, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Monocytes were cultured in 5% human serum in the presence or absence of troglitazone or 15 d-PGJ 2 as indicated. Supernatants of PMA-treated fibroblasts served as a control (Co). Gelatinolytic activity of freshly prepared monocytes is also shown (Mo). Equal amounts of lysates were loaded. Results shown were reproduced in three independent experiments. B: PPARγ activators decrease MMP-9 protein levels in supernatants from monocyte-derived macrophages (MØ). Vascular smooth muscle cells treated with PMA at 50 μg/L served as a control (Co). Supernatants from freshly prepared monocyte supernatant (Mo) are also shown. Similar results were reproduced in three independent experiments.

    Journal: The American Journal of Pathology

    Article Title: Macrophages in Human Atheroma Contain PPAR?

    doi:

    Figure Lengend Snippet: A: PPARγ activators decrease MMP-9 gelatinolytic activity in supernatants from monocyte-derived macrophages (MØ) in a concentration-dependent fashion, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Monocytes were cultured in 5% human serum in the presence or absence of troglitazone or 15 d-PGJ 2 as indicated. Supernatants of PMA-treated fibroblasts served as a control (Co). Gelatinolytic activity of freshly prepared monocytes is also shown (Mo). Equal amounts of lysates were loaded. Results shown were reproduced in three independent experiments. B: PPARγ activators decrease MMP-9 protein levels in supernatants from monocyte-derived macrophages (MØ). Vascular smooth muscle cells treated with PMA at 50 μg/L served as a control (Co). Supernatants from freshly prepared monocyte supernatant (Mo) are also shown. Similar results were reproduced in three independent experiments.

    Article Snippet: Processed samples were applied to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and protein was transferred to nitrocellulose membranes (Millipore, Bedford, MA) using semidry blotting for 1 hour, as described previously.

    Techniques: Activity Assay, Derivative Assay, Concentration Assay, Polyacrylamide Gel Electrophoresis, Zymography, Cell Culture

    Protein analysis of M. tuberculosis. (A) SDS-PAGE analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.

    Journal: Journal of Bacteriology

    Article Title: Microaerophilic Induction of the Alpha-Crystallin Chaperone Protein Homologue (hspX) mRNA of Mycobacterium tuberculosis

    doi: 10.1128/JB.183.18.5311-5316.2001

    Figure Lengend Snippet: Protein analysis of M. tuberculosis. (A) SDS-PAGE analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.

    Article Snippet: Twenty micrograms of protein for each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Tris-tricine 16.5% polyacrylamide gel (Bio-Rad).

    Techniques: SDS Page, Staining, Incubation

    Effects of IXD extract on the morphology of submandibular glands and in salivary total protein expression. Notes: ( A ) Hematoxylin and eosin staining was performed on paraffin-embedded submandibular gland tissues from normal and diabetic rats, either treated with water or IXD extract. Magnification=20×, scale bar=100 μm. ( B ) CBB staining showing total protein expression in the saliva of control and diabetic rats, either treated with water or IXD extract. The same volume of saliva was loaded in each well of a 10% SDS-PA gel, and the gel was stained with CBB. The black arrow indicates the molecular size of amylase (55 kDa) present in the saliva. Abbreviations: CBB, Coomassie Brilliant Blue; IXD, Ixeris dentata ; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Journal: Journal of Experimental Pharmacology

    Article Title: Ixeris dentata extract regulates salivary secretion through the activation of aquaporin-5 and prevents diabetes-induced xerostomia

    doi: 10.2147/JEP.S141807

    Figure Lengend Snippet: Effects of IXD extract on the morphology of submandibular glands and in salivary total protein expression. Notes: ( A ) Hematoxylin and eosin staining was performed on paraffin-embedded submandibular gland tissues from normal and diabetic rats, either treated with water or IXD extract. Magnification=20×, scale bar=100 μm. ( B ) CBB staining showing total protein expression in the saliva of control and diabetic rats, either treated with water or IXD extract. The same volume of saliva was loaded in each well of a 10% SDS-PA gel, and the gel was stained with CBB. The black arrow indicates the molecular size of amylase (55 kDa) present in the saliva. Abbreviations: CBB, Coomassie Brilliant Blue; IXD, Ixeris dentata ; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Article Snippet: Thirty µg of total protein were loaded per lane, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; BioRad), and transferred to a polyvinylidene fluoride membrane.

    Techniques: Expressing, Staining, SDS Page, Polyacrylamide Gel Electrophoresis

    Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae *

    doi: 10.1074/jbc.M114.633131

    Figure Lengend Snippet: Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.

    Article Snippet: Protein samples incubated with SDS sample buffer (50 m m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 1.33% β-mercaptoethanol) were separated on 12% Tris-glycine SDS-polyacrylamide gels by electrophoresis ( ) followed by transfer to Immobilon-P PVDF membranes (Millipore) at 100 V for 1.5 h. Membranes were then blocked overnight in 3% nonfat milk, phosphate-buffered saline (140.7 m m NaCl, 9.3 m m Na2 HPO4 , pH 7.4), 0.1% Tween 20.

    Techniques: Purification, Affinity Purification, SDS Page

    CNAP-tagged Coq proteins co-precipitate several other Coq proteins. W303, CNAP3, CNAP6, and CNAP9 purified mitochondria (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification with Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting with antisera to the designated yeast polypeptides. 25 μg of mitochondria protein were analyzed for each strain ( M ), and 2.5% of the first anti-PC elution ( E1 ) volume was loaded per strain (25 μl). Arrows denote each tagged protein in their respective blots. The predominant band in the Coq3 blot detected in the mitochondrial samples represents a background protein and not Coq3, accounting for its presence in CNAP3 mitochondria.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae *

    doi: 10.1074/jbc.M114.633131

    Figure Lengend Snippet: CNAP-tagged Coq proteins co-precipitate several other Coq proteins. W303, CNAP3, CNAP6, and CNAP9 purified mitochondria (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification with Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting with antisera to the designated yeast polypeptides. 25 μg of mitochondria protein were analyzed for each strain ( M ), and 2.5% of the first anti-PC elution ( E1 ) volume was loaded per strain (25 μl). Arrows denote each tagged protein in their respective blots. The predominant band in the Coq3 blot detected in the mitochondrial samples represents a background protein and not Coq3, accounting for its presence in CNAP3 mitochondria.

    Article Snippet: Protein samples incubated with SDS sample buffer (50 m m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 1.33% β-mercaptoethanol) were separated on 12% Tris-glycine SDS-polyacrylamide gels by electrophoresis ( ) followed by transfer to Immobilon-P PVDF membranes (Millipore) at 100 V for 1.5 h. Membranes were then blocked overnight in 3% nonfat milk, phosphate-buffered saline (140.7 m m NaCl, 9.3 m m Na2 HPO4 , pH 7.4), 0.1% Tween 20.

    Techniques: Purification, Affinity Purification, SDS Page

    Multiplication of parental and reassortant viruses in E-11 cells. (A) E-11 cells were transfected with a mixture of in vitro transcripts from pSJ1TK19 and pSJ2TK30 (a), pSG1TK5 and pSG2TK13 (b), pSJ1TK19 and pSG2TK13 (c), or pSG1TK5 and pSJ2TK30 (d). At 24 h posttransfection, viruses were detected by immunofluorescence staining with anti-SJNNV rabbit polyclonal antibody. (e) Mock-inoculated cells. Bar, 50 μm. (B) For each treatment, proteins were extracted from the E-11 cells (approximately 30,000 cells) at 24 h posttransfection with in vitro transcripts. Extracted proteins were separated by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. SJNNV and SGNNV CPs in the transfected cells were detected by Western blot analysis using anti-SJNNV rabbit polyclonal antibody. Authentic SJNNV and SGNNV virion samples were also used as positive controls.

    Journal: Journal of Virology

    Article Title: Identification of Host-Specificity Determinants in Betanodaviruses by Using Reassortants between Striped Jack Nervous Necrosis Virus and Sevenband Grouper Nervous Necrosis Virus

    doi: 10.1128/JVI.78.3.1256-1262.2004

    Figure Lengend Snippet: Multiplication of parental and reassortant viruses in E-11 cells. (A) E-11 cells were transfected with a mixture of in vitro transcripts from pSJ1TK19 and pSJ2TK30 (a), pSG1TK5 and pSG2TK13 (b), pSJ1TK19 and pSG2TK13 (c), or pSG1TK5 and pSJ2TK30 (d). At 24 h posttransfection, viruses were detected by immunofluorescence staining with anti-SJNNV rabbit polyclonal antibody. (e) Mock-inoculated cells. Bar, 50 μm. (B) For each treatment, proteins were extracted from the E-11 cells (approximately 30,000 cells) at 24 h posttransfection with in vitro transcripts. Extracted proteins were separated by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. SJNNV and SGNNV CPs in the transfected cells were detected by Western blot analysis using anti-SJNNV rabbit polyclonal antibody. Authentic SJNNV and SGNNV virion samples were also used as positive controls.

    Article Snippet: Western blot analysis was carried out as described previously , using an Immobilon-P transfer membrane (Millipore, Bedford, Mass.).

    Techniques: Transfection, In Vitro, Immunofluorescence, Staining, Polyacrylamide Gel Electrophoresis, Western Blot

    Two-dimensional gel electrophoresis patterns of the proteins-secreted into medium from  S. enterica  serovar Typhimurium CS2033 ( fljB ::Tn 10 ) (A), CS2034 ( fljB ::Tn 10 clpP ::Cm r ) (B), CS2144 ( fljB ::Tn 10 flhD-lac ) (C), and CS2145 ( fljB ::Tn 10 flhD-lac clpP ::Cm r ) (D). Protein spots excised for mass spectrometry analysis or transferred to the polyvinylidene difluoride membrane for amino-terminal sequence analysis are interpreted in the text.

    Journal: Journal of Bacteriology

    Article Title: The ClpXP ATP-Dependent Protease Regulates Flagellum Synthesis in Salmonella enterica Serovar Typhimurium

    doi: 10.1128/JB.184.3.645-653.2002

    Figure Lengend Snippet: Two-dimensional gel electrophoresis patterns of the proteins-secreted into medium from S. enterica serovar Typhimurium CS2033 ( fljB ::Tn 10 ) (A), CS2034 ( fljB ::Tn 10 clpP ::Cm r ) (B), CS2144 ( fljB ::Tn 10 flhD-lac ) (C), and CS2145 ( fljB ::Tn 10 flhD-lac clpP ::Cm r ) (D). Protein spots excised for mass spectrometry analysis or transferred to the polyvinylidene difluoride membrane for amino-terminal sequence analysis are interpreted in the text.

    Article Snippet: For immunoblot analysis, proteins separated on the SDS-12% polyacrylamide gels were transferred after electrophoresis onto Immobilon-P (Millipore) as previously reported ( ).

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Mass Spectrometry, Sequencing

    Distribution of tubulin in MDCK clone II/G and II/J cells after treatment with nocodazole. (A) Timeline scheme of experimental protocol used to disrupt microtubules. Confluent MDCK clone II/G and II/J cultures were established as described in MATERIALS AND METHODS. (B) After incubation in either the absence (−noc) or presence (+noc) of nocodazole, 120-h differentiated MDCK clone II/G and clone II/J cells were fixed and processed for immunofluorescence with monoclonal antibodies against α- and β-tubulin (see MATERIALS AND METHODS) to show the distribution of intact microtubules. Image stacks were produced from serial images collected for each specimen using a z step of 0.2 μm. (C) At the indicated times after the induction of cell–cell contacts, the amount of soluble (S) and polymerized tubulin (P) was determined in both clones of MDCK cells incubated in either the absence (−noc) or presence (+noc) of nocodazole. Samples were extracted in microtubule-stabilizing buffer containing 0.1% Triton X-100 and centrifuged at 20,000 × g to separate soluble (S) and insoluble fractions (P). The insoluble fractions were then solubilized in 0.5% Triton X-100. Equal amounts of supernatant and pellet fractions for each sample were separated by SDS-PAGE. Gels were then electrophoretically transferred to Immobilon polyvinylidene fluoride membrane and probed with a monoclonal antibody to β-tubulin. The β-tubulin antibody was visualized with a horseradish peroxidase-conjugated secondary antibody followed by enhanced chemiluminescence. Representative samples from two to four independent experiments for each time point are presented. Bar, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

    doi:

    Figure Lengend Snippet: Distribution of tubulin in MDCK clone II/G and II/J cells after treatment with nocodazole. (A) Timeline scheme of experimental protocol used to disrupt microtubules. Confluent MDCK clone II/G and II/J cultures were established as described in MATERIALS AND METHODS. (B) After incubation in either the absence (−noc) or presence (+noc) of nocodazole, 120-h differentiated MDCK clone II/G and clone II/J cells were fixed and processed for immunofluorescence with monoclonal antibodies against α- and β-tubulin (see MATERIALS AND METHODS) to show the distribution of intact microtubules. Image stacks were produced from serial images collected for each specimen using a z step of 0.2 μm. (C) At the indicated times after the induction of cell–cell contacts, the amount of soluble (S) and polymerized tubulin (P) was determined in both clones of MDCK cells incubated in either the absence (−noc) or presence (+noc) of nocodazole. Samples were extracted in microtubule-stabilizing buffer containing 0.1% Triton X-100 and centrifuged at 20,000 × g to separate soluble (S) and insoluble fractions (P). The insoluble fractions were then solubilized in 0.5% Triton X-100. Equal amounts of supernatant and pellet fractions for each sample were separated by SDS-PAGE. Gels were then electrophoretically transferred to Immobilon polyvinylidene fluoride membrane and probed with a monoclonal antibody to β-tubulin. The β-tubulin antibody was visualized with a horseradish peroxidase-conjugated secondary antibody followed by enhanced chemiluminescence. Representative samples from two to four independent experiments for each time point are presented. Bar, 10 μm.

    Article Snippet: For tubulin samples, the gels were electrophoretically transferred to Immobilon polyvinylidene fluoride membrane (Millipore, Bedford, MA).

    Techniques: Incubation, Immunofluorescence, Produced, Clone Assay, SDS Page

    Effect of E3-6.7K on the induction of procaspase-3 processing and PARP cleavage during TNF-induced apoptosis in vivo. Cell extracts were obtained from U937-neo and U937-6.7K cells that had been treated with 10 ng of TNF and 0.5 μg of CHX per ml for various lengths of time. Lysates containing equivalent amounts of protein based on the bicinchoninic acid (Pierce) protein concentration assay were loaded in each lane. After electrophoresis and transfer to PVDF membranes, blots were incubated with anti-caspase-3 rabbit antiserum that recognizes the 17- and 11-kDa subunits of the active, processed protein (A) and anti-PARP mouse monoclonal antibody that recognizes both the active 116-kDa and inactive 85-kDa forms of the protein (B). The blots were developed with a secondary antibody and visualized by chemiluminescence (Pierce Chemical). Similar results were obtained in two repeat experiments.

    Journal: Journal of Virology

    Article Title: Adenovirus E3-6.7K Maintains Calcium Homeostasis and Prevents Apoptosis and Arachidonic Acid Release

    doi: 10.1128/JVI.76.4.1578-1587.2002

    Figure Lengend Snippet: Effect of E3-6.7K on the induction of procaspase-3 processing and PARP cleavage during TNF-induced apoptosis in vivo. Cell extracts were obtained from U937-neo and U937-6.7K cells that had been treated with 10 ng of TNF and 0.5 μg of CHX per ml for various lengths of time. Lysates containing equivalent amounts of protein based on the bicinchoninic acid (Pierce) protein concentration assay were loaded in each lane. After electrophoresis and transfer to PVDF membranes, blots were incubated with anti-caspase-3 rabbit antiserum that recognizes the 17- and 11-kDa subunits of the active, processed protein (A) and anti-PARP mouse monoclonal antibody that recognizes both the active 116-kDa and inactive 85-kDa forms of the protein (B). The blots were developed with a secondary antibody and visualized by chemiluminescence (Pierce Chemical). Similar results were obtained in two repeat experiments.

    Article Snippet: Equivalent amounts of protein from each sample were resolved on a 10% glycine-SDS-polyacrylamide gel electrophoresis (PAGE) Laemmli gel system, blotted with the Towbin vertical-transfer wet system onto a 0.45-μm-pore-size Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore), and incubated with anti-PARP mouse monoclonal antibody (1:5,000 dilution; Pharmingen).

    Techniques: In Vivo, Protein Concentration, Electrophoresis, Incubation

    PKCε levels in DRG from CD and ED rats. A , Representative blot of PKC immunoreactivity in DRG samples from CD and ED rats (after 10 weeks of ethanol administration). Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with PKCε-specific antibody. B , Mean ± SEM for data showed a statistically significant difference between control diet-fed ( hatched bar ; n = 10) and ethanol-fed ( filled bar ; n = 11) rats. * p

    Journal: The Journal of Neuroscience

    Article Title: Key Role for the Epsilon Isoform of Protein Kinase C in Painful Alcoholic Neuropathy in the Rat

    doi: 10.1523/JNEUROSCI.20-22-08614.2000

    Figure Lengend Snippet: PKCε levels in DRG from CD and ED rats. A , Representative blot of PKC immunoreactivity in DRG samples from CD and ED rats (after 10 weeks of ethanol administration). Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with PKCε-specific antibody. B , Mean ± SEM for data showed a statistically significant difference between control diet-fed ( hatched bar ; n = 10) and ethanol-fed ( filled bar ; n = 11) rats. * p

    Article Snippet: Protein samples (8.3 μg/lane) were separated by SDS-PAGE on 8% minigels and thereafter transferred to polyvinylidene difluoride (PVDF) transfer membranes (Immobilon-P; Millipore, Bedford, MA) for 1 hr at 100 V. The membranes were washed briefly with TBS (20 m m Tris-HCl, pH 7.6, and 150 mm NaCl) and then blocked for 1 hr in Blotto [5% nonfat dry milk in TBS containing 0.05% Tween 20 (TBS-T)].

    Techniques: SDS Page

    Western blots probed with anti-lubricin mouse monoclonal antibodies (mAbs) 9g3, 7h12, 5c11, 6a8, and 8e3 detect lubricin in synovial fluid but do not detect other mucins. (A) Five μg of human lubricin purified from synovial fluid (PHL), and 1 μl of synovial fluid recovered from patients with osteoarthritis (OA), CACP, or from bovine, porcine, goat, dog, rat and guinea pig were electrophoresed under reducing conditions on 4–20% SDS-PAGE gradient gels, transferred to PVDF, and immunodetected using the biotin labeled mAbs. Note that the mAbs detect protein in the synovial fluid from patients with OA and from other species, but not from the patient with CACP, who is genetically deficient for lubricin. (B) 0.7 μg of Purified human lubricin (PHL), porcine gastric mucin (PM) and bovine submaxillary mucin (BSM) were electrophoresed under non-reducing conditions on 4–12% SDS-PAGE gradient gels, transferred to nitrocellulose and immunodetected using the mAbs. For mAb-8e3, 1.4 μg of PHL was loaded. Note that anti-lubricin mAbs did not cross react with BSM or PM.

    Journal: PLoS ONE

    Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

    doi: 10.1371/journal.pone.0116237

    Figure Lengend Snippet: Western blots probed with anti-lubricin mouse monoclonal antibodies (mAbs) 9g3, 7h12, 5c11, 6a8, and 8e3 detect lubricin in synovial fluid but do not detect other mucins. (A) Five μg of human lubricin purified from synovial fluid (PHL), and 1 μl of synovial fluid recovered from patients with osteoarthritis (OA), CACP, or from bovine, porcine, goat, dog, rat and guinea pig were electrophoresed under reducing conditions on 4–20% SDS-PAGE gradient gels, transferred to PVDF, and immunodetected using the biotin labeled mAbs. Note that the mAbs detect protein in the synovial fluid from patients with OA and from other species, but not from the patient with CACP, who is genetically deficient for lubricin. (B) 0.7 μg of Purified human lubricin (PHL), porcine gastric mucin (PM) and bovine submaxillary mucin (BSM) were electrophoresed under non-reducing conditions on 4–12% SDS-PAGE gradient gels, transferred to nitrocellulose and immunodetected using the mAbs. For mAb-8e3, 1.4 μg of PHL was loaded. Note that anti-lubricin mAbs did not cross react with BSM or PM.

    Article Snippet: The digests were mixed with 100 μl of 2X SDS-PAGE loading buffer containing 2% v/v BME and boiled for 5 min. Two μl were then electrophoresed in 4–20% gradient gels (Bio-Rad, Hercules, CA) and transferred to Immobilon P-PVDF (Millipore, Bedford, MA).

    Techniques: Western Blot, Purification, SDS Page, Labeling

    Immunoprecipitation of lubricin from human serum, plasma and synovial fluid. Biotin-labeled mAb-7h12 was mixed with plasma samples from a patient with CACP (CA697–1) or his unaffected father (CA697–2), serum samples from a patient with rheumatoid arthritis (RA) or an age- and sex-matched healthy control (Control), synovial fluid samples from a patient with osteoarthritis (OA), rheumatoid arthritis (RA), or CACP (CA), and with human (hLub-IP) and bovine (bLub-IP) lubricin that had been purified from synovial fluid. Antibody was recovered using streptavidin beads and proteins co-precipitated with mAb-7h12 were eluted, subjected to SDS-PAGE, transferred to PVDF, and immunodetected with mAb-7h12 (upper panel) or polyclonal Ab-J108N (lower panel). Note mAb-7h12 immunoprecipitated lubricin from plasma and serum that is also recognized by polyclonal antibody J108N. In contrast, lubricin immunoprecipitated from OA and RA synovial fluid is not recognized by polyclonal antibody J108N. J108N also does not recognize immunoprecipitated purified human lubricin (hLub-IP). The is no non-specific binding of lubricin to streptavidin beads as demonstrated by the lack of immunodetectable protein when mAb-7h12 is not used in the co-IP (no Ab control) of plasma from the unaffected CACP parent or synovial fluid from a patient with RA. As positive controls for immunodetection purified human and bovine lubricin (hLub and bLub) were directly subjected to SDS-PAGE for immunodetection.

    Journal: PLoS ONE

    Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

    doi: 10.1371/journal.pone.0116237

    Figure Lengend Snippet: Immunoprecipitation of lubricin from human serum, plasma and synovial fluid. Biotin-labeled mAb-7h12 was mixed with plasma samples from a patient with CACP (CA697–1) or his unaffected father (CA697–2), serum samples from a patient with rheumatoid arthritis (RA) or an age- and sex-matched healthy control (Control), synovial fluid samples from a patient with osteoarthritis (OA), rheumatoid arthritis (RA), or CACP (CA), and with human (hLub-IP) and bovine (bLub-IP) lubricin that had been purified from synovial fluid. Antibody was recovered using streptavidin beads and proteins co-precipitated with mAb-7h12 were eluted, subjected to SDS-PAGE, transferred to PVDF, and immunodetected with mAb-7h12 (upper panel) or polyclonal Ab-J108N (lower panel). Note mAb-7h12 immunoprecipitated lubricin from plasma and serum that is also recognized by polyclonal antibody J108N. In contrast, lubricin immunoprecipitated from OA and RA synovial fluid is not recognized by polyclonal antibody J108N. J108N also does not recognize immunoprecipitated purified human lubricin (hLub-IP). The is no non-specific binding of lubricin to streptavidin beads as demonstrated by the lack of immunodetectable protein when mAb-7h12 is not used in the co-IP (no Ab control) of plasma from the unaffected CACP parent or synovial fluid from a patient with RA. As positive controls for immunodetection purified human and bovine lubricin (hLub and bLub) were directly subjected to SDS-PAGE for immunodetection.

    Article Snippet: The digests were mixed with 100 μl of 2X SDS-PAGE loading buffer containing 2% v/v BME and boiled for 5 min. Two μl were then electrophoresed in 4–20% gradient gels (Bio-Rad, Hercules, CA) and transferred to Immobilon P-PVDF (Millipore, Bedford, MA).

    Techniques: Immunoprecipitation, Labeling, Purification, SDS Page, Binding Assay, Co-Immunoprecipitation Assay, Immunodetection

    Expression of human alpha-dystrobrevin isoforms in HL-60 cells undergoing granulocytic differentiation. (A) Cytosolic and nuclear proteins were isolated from proliferating and differentiating (0.5–96 h) HL-60 cells as described. The proteins were fractionated by SDS-PAGE on a 7–15% acrylamide gel gradient and transferred on to Immobilon PVDF membrane. The membranes were analyzed with polyclonal antibodies against human alpha-dystrobrevin and developed with an enhanced chemiluminescence detection system. (B) Total nuclear proteins were isolated from proliferating and differentiated (RA for 30 min, 24 h, and 96 h) HL-60 cells. The isolated proteins were fractionated by 2-DE, blotted on PVDF membranes, and analyzed with polyclonal antibodies against human alpha-dystrobrevin-gamma. The numerals I–IV denote different isoforms of human alpha-dystrobrevin detected in the nucleus of the HL-60 cells. Migration of the molecular size marker proteins is indicated to the right (kilodalton values).

    Journal: Molecular Biology of the Cell

    Article Title: Characterization of Human Alpha-Dystrobrevin Isoforms in HL-60 Human Promyelocytic Leukemia Cells Undergoing Granulocytic Differentiation

    doi: 10.1091/mbc.E02-03-0128

    Figure Lengend Snippet: Expression of human alpha-dystrobrevin isoforms in HL-60 cells undergoing granulocytic differentiation. (A) Cytosolic and nuclear proteins were isolated from proliferating and differentiating (0.5–96 h) HL-60 cells as described. The proteins were fractionated by SDS-PAGE on a 7–15% acrylamide gel gradient and transferred on to Immobilon PVDF membrane. The membranes were analyzed with polyclonal antibodies against human alpha-dystrobrevin and developed with an enhanced chemiluminescence detection system. (B) Total nuclear proteins were isolated from proliferating and differentiated (RA for 30 min, 24 h, and 96 h) HL-60 cells. The isolated proteins were fractionated by 2-DE, blotted on PVDF membranes, and analyzed with polyclonal antibodies against human alpha-dystrobrevin-gamma. The numerals I–IV denote different isoforms of human alpha-dystrobrevin detected in the nucleus of the HL-60 cells. Migration of the molecular size marker proteins is indicated to the right (kilodalton values).

    Article Snippet: After SDS-PAGE or 2-DE, proteins were transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) then blocked by incubating overnight at 4°C with 3% bovine serum albumin (BSA) dissolved in PBS supplemented with 0.18% Tween 20.

    Techniques: Expressing, Isolation, SDS Page, Acrylamide Gel Assay, Migration, Marker

    New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates (SDS fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing SDS/PAGE and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates (SDS fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing SDS/PAGE and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Recombinant, SDS Page, Western Blot, Microscopy, Immunofluorescence, Staining, Clone Assay, Sequencing

    Parkin transitions from a soluble to an aggregated state in adult human midbrain. (a) Representative Western blots of parkin, DJ-1, and LC3B distribution in human cortex, S. nigra (SN) and red nucleus (RN) brain specimens that had been serially fractionated into Tris-NaCl buffer-soluble (TS), Triton X-100-soluble (TX), 2% SDS-soluble (SDS) extracts and the pellet (P) lysed in 30% SDS-containing buffer. Lysates from PRKN -linked Parkinson disease (ARPD) brain and recombinant, human parkin (r-parkin) are included. (b-c) Relative distribution of parkin signal within each fraction for (b) cortex and (c) midbrain grouped by age ranges: young (Y; ≤ 20y; n=13); mid (M, > 20y,

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: Parkin transitions from a soluble to an aggregated state in adult human midbrain. (a) Representative Western blots of parkin, DJ-1, and LC3B distribution in human cortex, S. nigra (SN) and red nucleus (RN) brain specimens that had been serially fractionated into Tris-NaCl buffer-soluble (TS), Triton X-100-soluble (TX), 2% SDS-soluble (SDS) extracts and the pellet (P) lysed in 30% SDS-containing buffer. Lysates from PRKN -linked Parkinson disease (ARPD) brain and recombinant, human parkin (r-parkin) are included. (b-c) Relative distribution of parkin signal within each fraction for (b) cortex and (c) midbrain grouped by age ranges: young (Y; ≤ 20y; n=13); mid (M, > 20y,

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Western Blot, Recombinant

    Parkin’s secondary structure is altered by redox stress. (a) Silver staining of r-parkin in soluble (supernatant) and insoluble (pellet) phases following exposure to increasing concentrations of H 2 O 2 (0-2mM) and run under non-reducing conditions. Monomer (*) and high M r weight (HMW) r-parkin species are indicated. (b) Silver stained gel of r-parkin exposed to H 2 O 2 (10 mM), followed by treatment with increasing concentrations of DTT (0-100 mM) prior to centrifugation and loading of the supernatant onto SDS-PAGE. (c,d) Circular dichroism spectra of soluble, untreated r-parkin at (c) T=0 and (d) soluble (black line) and aggregated (red line) states following incubation at 37°C for T=5 days. The protein secondary structure shifts from a predominant appearance of α-helix dominated state, as demonstrated by the positive band at 193 nm and negative bands 208 nm (black lines), to the appearance of β-pleated sheet formation, as demonstrated by negative bands at 218 nm and a rise in molar ellipticity with positive bands at 195 nm (red lines) during spontaneous oxidation. (e-f) Quantitative analyses of IAA-modified cysteines captured by LC-MS/MS for (e) untreated vs. H 2 O 2 -exposed r-parkin, and (f) soluble compared to insoluble (pellet) fractions. Each data point represents the log2-transformed total IAA-signal intensities of single cysteine residues (n=3 runs for each). The cysteine pool is shown with the mean ± SEM; significance **p

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: Parkin’s secondary structure is altered by redox stress. (a) Silver staining of r-parkin in soluble (supernatant) and insoluble (pellet) phases following exposure to increasing concentrations of H 2 O 2 (0-2mM) and run under non-reducing conditions. Monomer (*) and high M r weight (HMW) r-parkin species are indicated. (b) Silver stained gel of r-parkin exposed to H 2 O 2 (10 mM), followed by treatment with increasing concentrations of DTT (0-100 mM) prior to centrifugation and loading of the supernatant onto SDS-PAGE. (c,d) Circular dichroism spectra of soluble, untreated r-parkin at (c) T=0 and (d) soluble (black line) and aggregated (red line) states following incubation at 37°C for T=5 days. The protein secondary structure shifts from a predominant appearance of α-helix dominated state, as demonstrated by the positive band at 193 nm and negative bands 208 nm (black lines), to the appearance of β-pleated sheet formation, as demonstrated by negative bands at 218 nm and a rise in molar ellipticity with positive bands at 195 nm (red lines) during spontaneous oxidation. (e-f) Quantitative analyses of IAA-modified cysteines captured by LC-MS/MS for (e) untreated vs. H 2 O 2 -exposed r-parkin, and (f) soluble compared to insoluble (pellet) fractions. Each data point represents the log2-transformed total IAA-signal intensities of single cysteine residues (n=3 runs for each). The cysteine pool is shown with the mean ± SEM; significance **p

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Silver Staining, Staining, Centrifugation, SDS Page, Incubation, Modification, Liquid Chromatography with Mass Spectroscopy, Transformation Assay

    Redox potentials of the cysteine pairs in the P1 and P2 loops of α-DsbB. ( A ) Redox equilibria of α-DsbB [CCSS] and α-DsbB [SSCC]. After equilibration in redox buffers of varying [DTTred]/[DTTox] ratios for 24 h, samples were treated with 10% TCA and free cysteines were labeled with AMS. Reduced and oxidized proteins were separated by 12% SDS-PAGE under non-reducing conditions. ( B ) Redox potentials of α-DsbB [CCSS] and α-DsbB [SSCC]. The fractions of α-DsbB [CCSS] or α-DsbB [SSCC] in the reduced state were calculated using ImageJ [36] and equilibrium constants were plotted against the fraction of the reduced state in PRISM® using the one phase decay option. The data presented are the standard mean and error from duplicate measurements.

    Journal: PLoS ONE

    Article Title: The ?-Proteobacteria Wolbachia pipientis Protein Disulfide Machinery Has a Regulatory Mechanism Absent in γ-Proteobacteria

    doi: 10.1371/journal.pone.0081440

    Figure Lengend Snippet: Redox potentials of the cysteine pairs in the P1 and P2 loops of α-DsbB. ( A ) Redox equilibria of α-DsbB [CCSS] and α-DsbB [SSCC]. After equilibration in redox buffers of varying [DTTred]/[DTTox] ratios for 24 h, samples were treated with 10% TCA and free cysteines were labeled with AMS. Reduced and oxidized proteins were separated by 12% SDS-PAGE under non-reducing conditions. ( B ) Redox potentials of α-DsbB [CCSS] and α-DsbB [SSCC]. The fractions of α-DsbB [CCSS] or α-DsbB [SSCC] in the reduced state were calculated using ImageJ [36] and equilibrium constants were plotted against the fraction of the reduced state in PRISM® using the one phase decay option. The data presented are the standard mean and error from duplicate measurements.

    Article Snippet: To identify protein-containing fractions, 15 µL samples from peak fractions were applied to a 4–12% Bis-Tris SDS PAGE (Life Technologies, USA).

    Techniques: Labeling, Affinity Magnetic Separation, SDS Page

    Immunodetection of Cr LPAAT 1 expression in C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The SDS ‐ PAGE gel was stained with blue dye (ProSieve EX Safe Stain— LONZA ) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS ‐ PAGE gel, and expression of Cr LPAAT 1 was detected using anti‐ HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE : pLM 21‐Cr LPAAT 1‐ HA overexpressors; three transformants ( OE 1, OE 2 and OE 3) are shown.

    Journal: Plant Biotechnology Journal

    Article Title: Identification of a Chlamydomonas plastidial 2‐lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content

    doi: 10.1111/pbi.12572

    Figure Lengend Snippet: Immunodetection of Cr LPAAT 1 expression in C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The SDS ‐ PAGE gel was stained with blue dye (ProSieve EX Safe Stain— LONZA ) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS ‐ PAGE gel, and expression of Cr LPAAT 1 was detected using anti‐ HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE : pLM 21‐Cr LPAAT 1‐ HA overexpressors; three transformants ( OE 1, OE 2 and OE 3) are shown.

    Article Snippet: Briefly, around 20 μg of protein was loaded onto a 12% Bis Tris SDS‐PAGE gel (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Immunodetection, Expressing, SDS Page, Staining

    Expression profiles of four DNA polymerases in Sulfolobus islandicus . Exponential growth cultures (A600 = 0.2) of the wild-type (WT) strain, S. islandicus E233S was treated with DNA damage agent (2 μM of NQO) or untreated (CK), and these cultures were then grown for 24 h during which cell samples were taken at indicated time points (hours) and used for preparation of cell extracts. Proteins in the cell extracts (10 μg) were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and DNA polymerases were identified by western blotting and hybridization using antibodies raised against each DNA polymerase. PCNA1, a subunit of the archaeal replication sliding clamp, serving as a loading control.

    Journal: Frontiers in Microbiology

    Article Title: A Unique B-Family DNA Polymerase Facilitating Error-Prone DNA Damage Tolerance in Crenarchaeota

    doi: 10.3389/fmicb.2020.01585

    Figure Lengend Snippet: Expression profiles of four DNA polymerases in Sulfolobus islandicus . Exponential growth cultures (A600 = 0.2) of the wild-type (WT) strain, S. islandicus E233S was treated with DNA damage agent (2 μM of NQO) or untreated (CK), and these cultures were then grown for 24 h during which cell samples were taken at indicated time points (hours) and used for preparation of cell extracts. Proteins in the cell extracts (10 μg) were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and DNA polymerases were identified by western blotting and hybridization using antibodies raised against each DNA polymerase. PCNA1, a subunit of the archaeal replication sliding clamp, serving as a loading control.

    Article Snippet: Proteins in each sample (10 μg) were separated according to their sizes on a 10% gel by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

    Techniques: Expressing, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Hybridization