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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 6526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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 18051 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sds polyacrylamide gel electrophoresis
    Detection of Apo-IV in chicken and quail plasma. Plasma lipoproteins with densities of ≤1.063 g/ml were <t>delipidated,</t> and 50 μg apolipoproteins from laying hen (lanes 1, 6, 11) or rooster (lanes 2, 7, 12), or 1 μl of whole plasma from laying hen (lanes 3, 8, 13), rooster (lanes 4, 9, 14), and quail rooster (lanes 5, 10, 15) were separated under reducing conditions on 12% <t>SDS-polyacrylamide</t> gels. After transfer to membranes, the blots in lanes 1–10 were incubated with rabbit anti-GST-Apo-IV antiserum (dilution 1:250) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μg/ml GST-Apo-IV, followed by peroxidase-conjugated goat anti-rabbit IgG. In lanes 11–15, preimmune rabbit serum was used instead of the anti-GST-Apo-IV rabbit antiserum.
    Sds Polyacrylamide Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sds polyacrylamide gels
    Tonic GnRH treatment increased <t>SRXN1</t> in L β T2 cells. Cultures of L β T2 cells were incubated for 24 h in DMEM/10% FBS, serum starved for 12 to 16 h, and then treated with tonic 10 nM GnRH for the indicated times, harvested, and subjected to <t>SDS-PAGE</t> and western blot analysis using antibodies against SRXN1 and β -actin. Band intensities were measured using quantitative chemiluminescence, and SRXN1 values were normalized to β -actin. (a) A representative blot from three independent experiments. (b) Mean values ± SEM normalized to time zero of four independent experiments. Different letters indicate a significant difference between groups ( P
    Sds Polyacrylamide Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 9802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher sds polyacrylamide gels
    Kap60 and Yar1 compete for Rps3 binding. ( a ) Kap60 co-elutes after Rps3-TAP purification. An RPS3 -TAP strain carrying a KAP60 -3xHA fusion was subjected to TAP purification. A KAP60 -3xHA strain without TAP-tag served as negative control (untagged). Lysates and final eluates (E-TAP) were analyzed by <t>SDS-PAGE</t> followed by <t>Coomassie</t> staining or Western blotting with the indicated antibodies. Note that there is some unspecific recovery of Kap60-3xHA in the eluate of the control strain; however, the protein is clearly enriched in the Rps3-TAP eluate. Kap123: Kap123 was detected in this band by mass spectrometry (Mascot score 211, 11 matched peptides, 12% sequence coverage). Kap60-3xHA: Kap60 was detected in this band by mass spectrometry (Mascot score 85, 4 matched peptides, 10% sequence coverage). ( b ) GST-tagged importins (Kap60ΔIBB or Kap123) were immobilized on glutathione-agarose beads. Truncated Kap60 lacking the N-terminal IBB domain (80 amino acids) was used in order to prevent inhibition of cargo binding by this domain in the absence of Kap95. Beads were incubated with purified, recombinant His6-Rps3/Flag-Yar1 complex or purified Flag-Yar1. Importins and bound material were eluted by boiling. Eluates were analyzed by SDS-PAGE and Coomassie staining or Western blotting with indicated antibodies. Asterisks in the Coomassie stained gel mark the respective importins, circles indicate bound Rps3. ( c ) Disruption of the Rps3-NLS impairs Kap60 binding. GST-tagged Kap60ΔIBB was incubated with His6-Rps3/Flag-Yar1 complex containing wild-type Rps3 or Rps3(K7/K10 > A) mutant protein. Kap60ΔIBB was eluted by TEV-cleavage. Eluates were analyzed by SDS-PAGE and Coomassie staining or Western blotting.
    Sds Polyacrylamide Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher sds polyacrylamide gel electrophoresis
    <t>SDS-PAGE</t> of the Proteus mirabilis strain ATCC 29245. The purified cytochrome b was loaded on 12.5% gel. The gel was stained with commassie brilliant blue after electrophoresis. Lane 1 (molecular mass marker proteins), lane 2 (sample 1, partially purified complex II after ion exchange chromatography) and lane 3, (sample 2, purified complex II). The molecular masses of three subunits of the Proteus mirabilis strain ATCC 29245 complex II were estimated to be 68, 28.5, and 19.5 kDa.
    Sds Polyacrylamide Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology sds polyacrylamide gel electrophoresis
    In vitro characterization of SecE(S105P) effects on pro-OmpA translocation. (A) Effects on different SecY mutational defects. IMVs were prepared from strains HM808 ( secY39 secE105 ) (lanes 1 and 2), GN04 ( secY39 ) (lanes 3 and 4), HM809 ( secY205 secE105 ) (lanes 5 and 6), GN05 ( secY205 ) (lanes 7 and 8), HM810 ( secY104 secE105 ) (lanes 9 and 10), GN09 ( secY104 ) (lanes 11 and 12), and TW156 ( secY + secE + ) (lanes 13 and 14). They were subjected to an in vitro translocation assay using 35 S-labeled pro-OmpA and wild-type SecA. The PMF was generated (+) or dissipated (−) by addition of 5 mM succinate or 10 μM carbonycyanide- m -chlorophenyl hydrazone, respectively. Reactions were allowed to proceed for 10 min at 37°C (upper panel) or 20°C (lower panel). Samples were treated with proteinase K and analyzed by <t>SDS-PAGE</t> and phosphorimager exposure. p, precursor forms; m, mature forms. (B) pro-OmpA translocation reaction mediated by the SecE(S105P) single-mutant IMV. IMVs were prepared from strains HM811 ( secE105 ) (lanes 1 to 10) and TW156 ( secE + ) (lanes 11 to 20). They were subjected to a 35 S-labeled pro-OmpA translocation assay at 20°C in the presence (+) or absence (−) of the PMF for the indicated lengths of time. Shown are SDS-PAGEprofiles after proteinase K treatment. i, fragments generated from incompletely translocated pro-OmpA. (C) Time courses of full-length translocation. Intensities of the full-length proOmpA product (mature and precursor forms) with a SecE(S105P) IMV (circles) or a Sec + IMV (triangles) are plotted against the reaction time. Open and solid symbols, results in the presence and absence of the PMF, respectively. (D) Time courses of generation of incompletely translocated products. Intensities of the incomplete translocation products (corresponding to the “i” bands in panel B) are plotted against the reaction time. Symbols are the same as in panel C.
    Sds Polyacrylamide Gel Electrophoresis, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology sds polyacrylamide gels
    Trx and TrxR expression and localization. (A) The levels of Trx and TrxR were examined in nuclear extracts (10 µg) from human <t>ERα-positive</t> breast (MCF-7) and ERα-negative breast (MDA-MB-231), U2 osteosarcoma (U2OS), and cervical (HeLa) cancer cells. (B) Trx and TrxR expression was examined in MCF-7 cells that had been treated with ethanol (−E 2 ) or 10 nM E 2 (+E 2 ) for 24 h. Samples were fractionated by <t>SDS-PAGE</t> and subjected to western analysis with a Trx- or TrxR-specific antibody. GAPDH levels were monitored to demonstrate that similar amounts of sample were loaded in each lane. (C) MCF-7 cells were treated with ethanol (−E 2 ) or 10 nM E 2 (+E 2 ) for 24 h. Expression of Trx and TrxR was monitored using immunocyto-chemistry with Trx- and TrxR-specific antibodies. DAPI counter-staining was used to detect cell nuclei. The insert in the upper right hand corner of the −E 2 .
    Sds Polyacrylamide Gels, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 803 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Schuell GmbH sds polyacrylamide gel electrophoresis
    Expression analysis of pUL69 and its betaherpesviral homologs. (A and B) HEK293T cells were transfected with plasmids encoding N-terminally FLAG-tagged (A) or Myc-tagged (B) versions of pUL69 (lane 2), pC69 (lane 3), pRh69 (lane 4), pM69 (lane 5), and pU42 of HHV6 (lane 6) and ElHV1 (lane 7), as indicated. Transfection of an empty vector served as a specificity control (mock, lanes 1). Cells were lysed under denaturing conditions at about 36 h posttransfection. Lysates were separated by <t>SDS-PAGE,</t> and protein expression was analyzed by Western blotting using anti-FLAG- or anti-Myc-specific antibodies. Molecular size markers are indicated (kDa).
    Sds Polyacrylamide Gel Electrophoresis, supplied by Schuell GmbH, used in various techniques. Bioz Stars score: 93/100, based on 768 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc sds polyacrylamide gel electrophoresis
    Cellular mRNA 3′-end processing enhances but is not required for PABPC-induced hyperadenylation. (A) HEK 293T cells were transfected with the indicated plasmids for 24 h, and then total RNA was isolated and Northern blotted with 32 P-labeled GFP and 18S probes. (B) Same protocol as described in the legend to panel A, but cells were treated with 5 ng/ml LMB for 12 h prior to RNA isolation to enhance detection of hyperadenylated species. (C) HEK 293T cells were transfected with the indicated plasmids for 24 h and then treated with 5 ng/ml LMB for 12 h. Total RNA was isolated and incubated in the presence or absence of oligo(dT) and then digested with RNaseH. RNA was visualized by Northern blotting with 32 P-labeled GFP and 18S probes. (D) HEK 293T cells were transfected twice over 48 h with either PAPII siRNAs or control siRNAs and then subsequently transfected in duplicate with DNA plasmids expressing GFP-A 60 -HR alone or together with Flag-PABPC1-NRS for 24 h. Samples were treated with 5 ng/ml LMB for 6 h prior to harvesting either protein (top) or RNA (bottom). Protein lysates were resolved by <t>SDS-PAGE</t> and immunoblotted with antibodies against PAPII, Flag, or actin (as a loading control). RNA from each sample was Northern blotted with 32 P-labeled GFP and 18S probes.
    Sds Polyacrylamide Gel Electrophoresis, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 843 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Membrane association of the rotavirus VP5 proteins. HEK293 cells were transfected with pcDNA4 constructs expressing VP5N248, VP5N248(394R), or VP5N265. Cellular membranes were fractionated by sucrose gradient centrifugation. His-tagged VP5N248, VP5N248(394R), or VP5N265 proteins were precipitated from total cell lysates (Total), membrane fractions (Membrane), or soluble fractions (Soluble) using Ni-NTA agarose. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted using an anti-HisG antibody.

    Journal: Journal of Virology

    Article Title: Discrete Domains within the Rotavirus VP5* Direct Peripheral Membrane Association and Membrane Permeability

    doi: 10.1128/JVI.78.4.2037-2044.2004

    Figure Lengend Snippet: Membrane association of the rotavirus VP5 proteins. HEK293 cells were transfected with pcDNA4 constructs expressing VP5N248, VP5N248(394R), or VP5N265. Cellular membranes were fractionated by sucrose gradient centrifugation. His-tagged VP5N248, VP5N248(394R), or VP5N265 proteins were precipitated from total cell lysates (Total), membrane fractions (Membrane), or soluble fractions (Soluble) using Ni-NTA agarose. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted using an anti-HisG antibody.

    Article Snippet: Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted using anti-HisG primary antibody (Invitrogen).

    Techniques: Transfection, Construct, Expressing, Gradient Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot

    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

    (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

    Detection of Apo-IV in chicken and quail plasma. Plasma lipoproteins with densities of ≤1.063 g/ml were delipidated, and 50 μg apolipoproteins from laying hen (lanes 1, 6, 11) or rooster (lanes 2, 7, 12), or 1 μl of whole plasma from laying hen (lanes 3, 8, 13), rooster (lanes 4, 9, 14), and quail rooster (lanes 5, 10, 15) were separated under reducing conditions on 12% SDS-polyacrylamide gels. After transfer to membranes, the blots in lanes 1–10 were incubated with rabbit anti-GST-Apo-IV antiserum (dilution 1:250) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μg/ml GST-Apo-IV, followed by peroxidase-conjugated goat anti-rabbit IgG. In lanes 11–15, preimmune rabbit serum was used instead of the anti-GST-Apo-IV rabbit antiserum.

    Journal: Biochimie

    Article Title: A novel estrogen-regulated avian apolipoprotein

    doi: 10.1016/j.biochi.2013.09.005

    Figure Lengend Snippet: Detection of Apo-IV in chicken and quail plasma. Plasma lipoproteins with densities of ≤1.063 g/ml were delipidated, and 50 μg apolipoproteins from laying hen (lanes 1, 6, 11) or rooster (lanes 2, 7, 12), or 1 μl of whole plasma from laying hen (lanes 3, 8, 13), rooster (lanes 4, 9, 14), and quail rooster (lanes 5, 10, 15) were separated under reducing conditions on 12% SDS-polyacrylamide gels. After transfer to membranes, the blots in lanes 1–10 were incubated with rabbit anti-GST-Apo-IV antiserum (dilution 1:250) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μg/ml GST-Apo-IV, followed by peroxidase-conjugated goat anti-rabbit IgG. In lanes 11–15, preimmune rabbit serum was used instead of the anti-GST-Apo-IV rabbit antiserum.

    Article Snippet: 2.4 Microsequencing The lipoprotein fraction of d ≤ 1.063 of rooster plasma was isolated by ultracentrifugal floatation, delipidated, the residue subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto a polyvinylidene difluoride membrane (Immobilon P, 0.45 mm, Millipore Corp., Bedford, MA).

    Techniques: Incubation

    Yolk VLDL does not harbor detectable amounts of Apo-IV. One μl of plasma from laying hen (LH) or rooster, or 50 μg delipidated VLDL isolated from egg yolk (yVLDL) were separated by 12% (A) or 15% (B) SDS-PAGE in the presence of 50 mM DTT. (A) Western blotting analysis of Apo-IV using mouse anti-GST-Apo-IV antiserum (dilution 1:1500), followed by peroxidase-conjugated rabbit anti-mouse IgG. (B) ApoVLDL-II was visualized by Western blot analysis using rabbit anti-apoVLDL-II antiserum (dilution 1:1500) and peroxidase-conjugated goat anti-rabbit IgG.

    Journal: Biochimie

    Article Title: A novel estrogen-regulated avian apolipoprotein

    doi: 10.1016/j.biochi.2013.09.005

    Figure Lengend Snippet: Yolk VLDL does not harbor detectable amounts of Apo-IV. One μl of plasma from laying hen (LH) or rooster, or 50 μg delipidated VLDL isolated from egg yolk (yVLDL) were separated by 12% (A) or 15% (B) SDS-PAGE in the presence of 50 mM DTT. (A) Western blotting analysis of Apo-IV using mouse anti-GST-Apo-IV antiserum (dilution 1:1500), followed by peroxidase-conjugated rabbit anti-mouse IgG. (B) ApoVLDL-II was visualized by Western blot analysis using rabbit anti-apoVLDL-II antiserum (dilution 1:1500) and peroxidase-conjugated goat anti-rabbit IgG.

    Article Snippet: 2.4 Microsequencing The lipoprotein fraction of d ≤ 1.063 of rooster plasma was isolated by ultracentrifugal floatation, delipidated, the residue subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto a polyvinylidene difluoride membrane (Immobilon P, 0.45 mm, Millipore Corp., Bedford, MA).

    Techniques: Isolation, SDS Page, Western Blot

    Lobeline treatment increases laminin binding. Lysates of surface biotinylated wild type ( wt ) ( A ) or mdx myotubes ( B ) treated with lobeline ( L ) or control ( C ) were separated by SDS-PAGE and blotted, laminin was incubated with the blots, and bound laminin was detected with anti-laminin. Blots were stripped and reprobed with mAb IIH6.

    Journal: The Journal of Biological Chemistry

    Article Title: High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding *

    doi: 10.1074/jbc.M111.334581

    Figure Lengend Snippet: Lobeline treatment increases laminin binding. Lysates of surface biotinylated wild type ( wt ) ( A ) or mdx myotubes ( B ) treated with lobeline ( L ) or control ( C ) were separated by SDS-PAGE and blotted, laminin was incubated with the blots, and bound laminin was detected with anti-laminin. Blots were stripped and reprobed with mAb IIH6.

    Article Snippet: Wild type and mdx muscle cell eluates were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore).

    Techniques: Binding Assay, SDS Page, Incubation

    Lobeline increases abundance of multiple muscle cell proteins. A , wild type ( wt ) primary myoblasts differentiated in the presence of 100 μ m lobeline ( L ) or control ( C ) were solubilized in radioimmune precipitation assay buffer, and 60 μg of protein was separated by SDS-PAGE and blotted to nitrocellulose. Membranes were probed with antibodies against agrin, dystrophin, utrophin, dystroglycans (α- and β-DG), sarcospan ( SSPN ), sarcoglycans (α-, β-, and γ-SG), β1D integrin, dysferlin, and caveolin-3. GAPDH was a loading control. Lobeline treatment increased agrin, integrin, dysferlin, DGC, and UGC protein levels. B , mdx primary myoblasts were treated as described in A . Membranes were probed similarly with the addition of antibody to β-SG. Dystrophin protein was not detected due to mutation in the dystrophin gene in mdx cells. Increases were observed for agrin, dysferlin, utrophin, and α- and β-dystroglycans.

    Journal: The Journal of Biological Chemistry

    Article Title: High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding *

    doi: 10.1074/jbc.M111.334581

    Figure Lengend Snippet: Lobeline increases abundance of multiple muscle cell proteins. A , wild type ( wt ) primary myoblasts differentiated in the presence of 100 μ m lobeline ( L ) or control ( C ) were solubilized in radioimmune precipitation assay buffer, and 60 μg of protein was separated by SDS-PAGE and blotted to nitrocellulose. Membranes were probed with antibodies against agrin, dystrophin, utrophin, dystroglycans (α- and β-DG), sarcospan ( SSPN ), sarcoglycans (α-, β-, and γ-SG), β1D integrin, dysferlin, and caveolin-3. GAPDH was a loading control. Lobeline treatment increased agrin, integrin, dysferlin, DGC, and UGC protein levels. B , mdx primary myoblasts were treated as described in A . Membranes were probed similarly with the addition of antibody to β-SG. Dystrophin protein was not detected due to mutation in the dystrophin gene in mdx cells. Increases were observed for agrin, dysferlin, utrophin, and α- and β-dystroglycans.

    Article Snippet: Wild type and mdx muscle cell eluates were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore).

    Techniques: SDS Page, Mutagenesis

    Identification of WFA binding glycoproteins in control-treated versus lobeline-treated cells. A , equal amounts of cell protein from lysates of C2C12 myotubes treated with 100 μ m lobeline ( L ) or control ( C ) for 48 h were precipitated with WFA beads, bound proteins were eluted with GalNAc, eluted proteins were separated by SDS-PAGE, and blots were probed with WFA or mAb IIH6. Equal amounts of cell protein from lysates of wild type ( wt ) ( B ) or mdx myotubes ( C ) treated with lobeline or control were precipitated with WFA beads, bound proteins were eluted with GalNAc, eluted proteins were separated by SDS-PAGE, and blots were probed with WFA or mAbs IIH6 or MANDAG2 ( MA ), which recognizes β-DG. Results are representative of four independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding *

    doi: 10.1074/jbc.M111.334581

    Figure Lengend Snippet: Identification of WFA binding glycoproteins in control-treated versus lobeline-treated cells. A , equal amounts of cell protein from lysates of C2C12 myotubes treated with 100 μ m lobeline ( L ) or control ( C ) for 48 h were precipitated with WFA beads, bound proteins were eluted with GalNAc, eluted proteins were separated by SDS-PAGE, and blots were probed with WFA or mAb IIH6. Equal amounts of cell protein from lysates of wild type ( wt ) ( B ) or mdx myotubes ( C ) treated with lobeline or control were precipitated with WFA beads, bound proteins were eluted with GalNAc, eluted proteins were separated by SDS-PAGE, and blots were probed with WFA or mAbs IIH6 or MANDAG2 ( MA ), which recognizes β-DG. Results are representative of four independent experiments.

    Article Snippet: Wild type and mdx muscle cell eluates were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore).

    Techniques: Binding Assay, SDS Page

    Secreted APOL1 does not contribute to cytotoxicity. A : APOL1-myc splice variant proteins are released into culture media of transfected HEK293T cells. At 5 h after transfection, cells were transferred into DMEM supplemented with 2% FCS. After overnight incubation, culture media were collected, filtered, and precleared with Protein A/G PLUS-agarose, and APOL1-myc was immunoprecipitated using anti-myc antibodies conjugated with agarose beads. Cell lysate proteins and immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting for expression of APOL1 and GAPDH. In addition to APOL1 proteins, nonspecific proteins (most likely derived from serum-containing media) were also detected in anti-myc immunoprecipitates (asterisk). B : intracellular APOL1 is refractory to extraction with the nonionic detergent saponin. Permeabilization of cell membranes with saponin depleted cytosolic GAPDH but retained actin. At 24 h after transfection, cells were permeabilized with 0.1% saponin for 10 min, and protein lysates from untreated (Control) or saponin-treated cells (+Saponin) were analyzed by immunoblotting for expression of APOL1, GAPDH, and actin. C : secreted APOL1 does not contribute to the intracellular pool of APOL1. Transfected cells were cultured in the presence of 80 μM dynasore or solvent (DMSO). After 24 h, cells were permeabilized with 0.1% saponin, and cell protein lysates were separated by SDS-PAGE and analyzed for expression of APOL1, GAPDH, and actin. D : secreted APOL1 does not contribute to cytotoxicity. Cells were transfected with cytotoxic and noncytotoxic APOL1 variants B1 and B3, respectively, and cultured in the absence or presence of 80 μM dynasore. After 24 h, cell culture media were collected and analyzed for released LDH ( top ). Values are means ± SD of 3 independent samples. Corresponding protein lysates were analyzed by immunoblotting for expression of APOL1 and actin ( bottom ). * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Exon 4-encoded sequence is a major determinant of cytotoxicity of apolipoprotein L1

    doi: 10.1152/ajpcell.00384.2014

    Figure Lengend Snippet: Secreted APOL1 does not contribute to cytotoxicity. A : APOL1-myc splice variant proteins are released into culture media of transfected HEK293T cells. At 5 h after transfection, cells were transferred into DMEM supplemented with 2% FCS. After overnight incubation, culture media were collected, filtered, and precleared with Protein A/G PLUS-agarose, and APOL1-myc was immunoprecipitated using anti-myc antibodies conjugated with agarose beads. Cell lysate proteins and immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting for expression of APOL1 and GAPDH. In addition to APOL1 proteins, nonspecific proteins (most likely derived from serum-containing media) were also detected in anti-myc immunoprecipitates (asterisk). B : intracellular APOL1 is refractory to extraction with the nonionic detergent saponin. Permeabilization of cell membranes with saponin depleted cytosolic GAPDH but retained actin. At 24 h after transfection, cells were permeabilized with 0.1% saponin for 10 min, and protein lysates from untreated (Control) or saponin-treated cells (+Saponin) were analyzed by immunoblotting for expression of APOL1, GAPDH, and actin. C : secreted APOL1 does not contribute to the intracellular pool of APOL1. Transfected cells were cultured in the presence of 80 μM dynasore or solvent (DMSO). After 24 h, cells were permeabilized with 0.1% saponin, and cell protein lysates were separated by SDS-PAGE and analyzed for expression of APOL1, GAPDH, and actin. D : secreted APOL1 does not contribute to cytotoxicity. Cells were transfected with cytotoxic and noncytotoxic APOL1 variants B1 and B3, respectively, and cultured in the absence or presence of 80 μM dynasore. After 24 h, cell culture media were collected and analyzed for released LDH ( top ). Values are means ± SD of 3 independent samples. Corresponding protein lysates were analyzed by immunoblotting for expression of APOL1 and actin ( bottom ). * P

    Article Snippet: After 24 h, the cells were transfected with pcDNA3.1 or APOL1 expression vector , and after an additional 24 h, cells were lysed in RIPA buffer (Santa Cruz Biotechnology), and an equal amount of lysate proteins (20–50 μg) was separated on 10% SDS-polyacrylamide gel and analyzed by immunoblotting for expression of APOL1 (HPA018885 antibody, Sigma-Aldrich), Atg5 (Cell Signaling), and actin (Sigma-Aldrich).

    Techniques: Variant Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Expressing, Derivative Assay, Cell Culture

    Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Activation Assay, SDS Page

    Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Software, Expressing, SDS Page, Western Blot, Standard Deviation

    Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Activation Assay, SDS Page, Standard Deviation

    Isolated collagen species from chicken and xenopus on 6% SDS-PAGE. A, chicken collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction ; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. B, xenopus collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. β11(I) and β12(I) are cross-linked α1-α1 and α1-α2 chain dimers, respectively.

    Journal: PLoS ONE

    Article Title: Insights on the Evolution of Prolyl 3-Hydroxylation Sites from Comparative Analysis of Chicken and Xenopus Fibrillar Collagens

    doi: 10.1371/journal.pone.0019336

    Figure Lengend Snippet: Isolated collagen species from chicken and xenopus on 6% SDS-PAGE. A, chicken collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction ; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. B, xenopus collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. β11(I) and β12(I) are cross-linked α1-α1 and α1-α2 chain dimers, respectively.

    Article Snippet: Protein samples were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue G-250 (Sigma-Aldrich) .

    Techniques: Isolation, SDS Page

    Target specificity of SB202190. HCL14 cells were pretreated with SB202190 in serum-free medium for 1 hour, mock-irradiated or irradiated with 250 kJ/m 2 UVA, and then continued in serum-free medium supplemented with SB202190 until each desired time point. Ten micrograms (MAPKAPK2) or 40 µg (phospho-JNK and phospho-ERK) of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody concentrations for the target proteins. Western blot data are representative of at least two independent experiments. (A) Effect of SB202190 on p38 activity through detection of changes in MAPKAPK2 phosphorylation; (B) effect of SB202190 on JNK activation; and (C) effect of SB202190 on ERK activation.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP-1 Activation and c-Fos Expression 1

    doi:

    Figure Lengend Snippet: Target specificity of SB202190. HCL14 cells were pretreated with SB202190 in serum-free medium for 1 hour, mock-irradiated or irradiated with 250 kJ/m 2 UVA, and then continued in serum-free medium supplemented with SB202190 until each desired time point. Ten micrograms (MAPKAPK2) or 40 µg (phospho-JNK and phospho-ERK) of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody concentrations for the target proteins. Western blot data are representative of at least two independent experiments. (A) Effect of SB202190 on p38 activity through detection of changes in MAPKAPK2 phosphorylation; (B) effect of SB202190 on JNK activation; and (C) effect of SB202190 on ERK activation.

    Article Snippet: Lysates (40 µg) were resolved on 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gels and were then transferred to Immobilon-P nylon membranes (Millipore, Bedford, MA).

    Techniques: Irradiation, Western Blot, Activity Assay, Activation Assay

    Target specificity of SP600125. HCL14 cells were pretreated with a SP600125 in serum-free medium for 1 hour, mock-irradiated or irradiated with 250 kJ/m 2 UVA, and then continued in serum-free medium supplemented with SP600125 until each desired time point. Forty micrograms of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody concentrations for the target proteins. Western blot data are representative of at least two independent experiments. (A) Effect of SP600125 on JNK activity as detected by changes in c-Jun phosphorylation and expression; (B) effect of SP600125 on p38 activation; and (C) effect of SP600125 on ERK activation.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP-1 Activation and c-Fos Expression 1

    doi:

    Figure Lengend Snippet: Target specificity of SP600125. HCL14 cells were pretreated with a SP600125 in serum-free medium for 1 hour, mock-irradiated or irradiated with 250 kJ/m 2 UVA, and then continued in serum-free medium supplemented with SP600125 until each desired time point. Forty micrograms of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody concentrations for the target proteins. Western blot data are representative of at least two independent experiments. (A) Effect of SP600125 on JNK activity as detected by changes in c-Jun phosphorylation and expression; (B) effect of SP600125 on p38 activation; and (C) effect of SP600125 on ERK activation.

    Article Snippet: Lysates (40 µg) were resolved on 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gels and were then transferred to Immobilon-P nylon membranes (Millipore, Bedford, MA).

    Techniques: Irradiation, Western Blot, Activity Assay, Expressing, Activation Assay

    Effects of SB202190 and SP600125 on UVA-induced c-Fos protein expression. HCL14 cells were pretreated with a MAPK inhibitor in serum-free medium for 1 hour, mock-irradiated or irradiated with 250 kJ/m 2 UVA, and then continued in serum-free medium supplemented with the respective inhibitor for 1 hour. Forty micrograms of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody concentrations for c-Fos. Western blot data are representative of two independent experiments. (A) SB202190; and (B) SP600125.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP-1 Activation and c-Fos Expression 1

    doi:

    Figure Lengend Snippet: Effects of SB202190 and SP600125 on UVA-induced c-Fos protein expression. HCL14 cells were pretreated with a MAPK inhibitor in serum-free medium for 1 hour, mock-irradiated or irradiated with 250 kJ/m 2 UVA, and then continued in serum-free medium supplemented with the respective inhibitor for 1 hour. Forty micrograms of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody concentrations for c-Fos. Western blot data are representative of two independent experiments. (A) SB202190; and (B) SP600125.

    Article Snippet: Lysates (40 µg) were resolved on 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gels and were then transferred to Immobilon-P nylon membranes (Millipore, Bedford, MA).

    Techniques: Expressing, Irradiation, Western Blot

    Western time course for UVA-induced MAPK activation. HCL14 cells were mock-irradiated or irradiated with 250 kJ/m 2 UVA and harvested at the appropriate time points postirradiation. Forty micrograms of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody concentrations for each MAPK. Western blot data are representative of at least two independent experiments. (A) p38; (B) JNK; and (C) ERK.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP-1 Activation and c-Fos Expression 1

    doi:

    Figure Lengend Snippet: Western time course for UVA-induced MAPK activation. HCL14 cells were mock-irradiated or irradiated with 250 kJ/m 2 UVA and harvested at the appropriate time points postirradiation. Forty micrograms of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody concentrations for each MAPK. Western blot data are representative of at least two independent experiments. (A) p38; (B) JNK; and (C) ERK.

    Article Snippet: Lysates (40 µg) were resolved on 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gels and were then transferred to Immobilon-P nylon membranes (Millipore, Bedford, MA).

    Techniques: Western Blot, Activation Assay, Irradiation

    Tonic GnRH treatment increased SRXN1 in L β T2 cells. Cultures of L β T2 cells were incubated for 24 h in DMEM/10% FBS, serum starved for 12 to 16 h, and then treated with tonic 10 nM GnRH for the indicated times, harvested, and subjected to SDS-PAGE and western blot analysis using antibodies against SRXN1 and β -actin. Band intensities were measured using quantitative chemiluminescence, and SRXN1 values were normalized to β -actin. (a) A representative blot from three independent experiments. (b) Mean values ± SEM normalized to time zero of four independent experiments. Different letters indicate a significant difference between groups ( P

    Journal: Endocrinology

    Article Title: SRXN1 Is Necessary for Resolution of GnRH-Induced Oxidative Stress and Induction of Gonadotropin Gene Expression

    doi: 10.1210/en.2019-00283

    Figure Lengend Snippet: Tonic GnRH treatment increased SRXN1 in L β T2 cells. Cultures of L β T2 cells were incubated for 24 h in DMEM/10% FBS, serum starved for 12 to 16 h, and then treated with tonic 10 nM GnRH for the indicated times, harvested, and subjected to SDS-PAGE and western blot analysis using antibodies against SRXN1 and β -actin. Band intensities were measured using quantitative chemiluminescence, and SRXN1 values were normalized to β -actin. (a) A representative blot from three independent experiments. (b) Mean values ± SEM normalized to time zero of four independent experiments. Different letters indicate a significant difference between groups ( P

    Article Snippet: Isolated proteins were separated by electrophoresis on 14% (for SRXN1) or 12% (for PRDX-SO2/3) SDS polyacrylamide gels (Mini-PROTEAN; Bio-Rad Laboratories), and transferred to nitrocellulose membranes.

    Techniques: Incubation, SDS Page, Western Blot

    DPI and NAC attenuated GnRH pulse–mediated SRXN1 induction. L β T2 cells were cultured for 24 h in DMEM/10% FBS and then serum starved for 12 to 16 h. Cultures were then preincubated with the NADPH oxidase inhibitor DPI at 5 μM or the reactive oxygen scavenger NAC at 20 mM for 30 min, and then stimulated with 10 nM GnRH for 4 h. Cells were harvested and subjected to SDS-PAGE and western blot analysis using antibodies against SRXN1 and β -actin. Intensity values were measured by quantitative chemiluminescence. (a) A representative blot from three independent experiments. (b) The chart represents mean values ± SEM normalized to vehicle (Veh) of four independent determinations. Both DPI and NAC inhibited GnRH-induced increase in SRXN1. Different letters indicate a significant difference between groups ( P

    Journal: Endocrinology

    Article Title: SRXN1 Is Necessary for Resolution of GnRH-Induced Oxidative Stress and Induction of Gonadotropin Gene Expression

    doi: 10.1210/en.2019-00283

    Figure Lengend Snippet: DPI and NAC attenuated GnRH pulse–mediated SRXN1 induction. L β T2 cells were cultured for 24 h in DMEM/10% FBS and then serum starved for 12 to 16 h. Cultures were then preincubated with the NADPH oxidase inhibitor DPI at 5 μM or the reactive oxygen scavenger NAC at 20 mM for 30 min, and then stimulated with 10 nM GnRH for 4 h. Cells were harvested and subjected to SDS-PAGE and western blot analysis using antibodies against SRXN1 and β -actin. Intensity values were measured by quantitative chemiluminescence. (a) A representative blot from three independent experiments. (b) The chart represents mean values ± SEM normalized to vehicle (Veh) of four independent determinations. Both DPI and NAC inhibited GnRH-induced increase in SRXN1. Different letters indicate a significant difference between groups ( P

    Article Snippet: Isolated proteins were separated by electrophoresis on 14% (for SRXN1) or 12% (for PRDX-SO2/3) SDS polyacrylamide gels (Mini-PROTEAN; Bio-Rad Laboratories), and transferred to nitrocellulose membranes.

    Techniques: Cell Culture, SDS Page, Western Blot

    MAPK1/3 inhibitors reduced the SRXN1 response to GnRH stimulation. Cultured L β T2 cells were incubated for 24 h in DMEM/10% FBS; serum starved for 12 to 16 hours; pretreated with vehicle (Veh; dimethyl sulfoxide), 10 µM SP600125 (SP), 20 µM SB202190 (SB), 30 µM PD98059 (PD), or 2 µM U0126 (U) for 30 min; and then subsequently treated with 10 nM GnRH for 4 h. Cells were lysed and subjected to SDS-PAGE and western blot analysis using antibodies against SRXN1 and β -actin. Band intensities were measured using quantitative chemiluminescence, and SRXN1 values were normalized to β -actin. (a) A representative blot from three independent experiments. The chart represents mean values ± SEM, normalized to Veh, of four independent determinations. Different letters indicate a significant difference between groups ( P

    Journal: Endocrinology

    Article Title: SRXN1 Is Necessary for Resolution of GnRH-Induced Oxidative Stress and Induction of Gonadotropin Gene Expression

    doi: 10.1210/en.2019-00283

    Figure Lengend Snippet: MAPK1/3 inhibitors reduced the SRXN1 response to GnRH stimulation. Cultured L β T2 cells were incubated for 24 h in DMEM/10% FBS; serum starved for 12 to 16 hours; pretreated with vehicle (Veh; dimethyl sulfoxide), 10 µM SP600125 (SP), 20 µM SB202190 (SB), 30 µM PD98059 (PD), or 2 µM U0126 (U) for 30 min; and then subsequently treated with 10 nM GnRH for 4 h. Cells were lysed and subjected to SDS-PAGE and western blot analysis using antibodies against SRXN1 and β -actin. Band intensities were measured using quantitative chemiluminescence, and SRXN1 values were normalized to β -actin. (a) A representative blot from three independent experiments. The chart represents mean values ± SEM, normalized to Veh, of four independent determinations. Different letters indicate a significant difference between groups ( P

    Article Snippet: Isolated proteins were separated by electrophoresis on 14% (for SRXN1) or 12% (for PRDX-SO2/3) SDS polyacrylamide gels (Mini-PROTEAN; Bio-Rad Laboratories), and transferred to nitrocellulose membranes.

    Techniques: Cell Culture, Incubation, SDS Page, Western Blot

    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

    Silver-stained SDS-PAGE of pRBC saponin extracts run through four affinity chromatography columns where AGMA1, ISA1, ISA23 or ARGO7 had been immobilized. ( A ) A RBC extract was first loaded, and after the corresponding washing-elution-washing steps ( B ) a pRBC extract was loaded in the same column. The approximate masses (kDa) of the seven bands from the molecular weight marker are indicated in the space between the gels.

    Journal: Pharmaceutics

    Article Title: Polyamidoamine Nanoparticles for the Oral Administration of Antimalarial Drugs

    doi: 10.3390/pharmaceutics10040225

    Figure Lengend Snippet: Silver-stained SDS-PAGE of pRBC saponin extracts run through four affinity chromatography columns where AGMA1, ISA1, ISA23 or ARGO7 had been immobilized. ( A ) A RBC extract was first loaded, and after the corresponding washing-elution-washing steps ( B ) a pRBC extract was loaded in the same column. The approximate masses (kDa) of the seven bands from the molecular weight marker are indicated in the space between the gels.

    Article Snippet: For SDS-polyacrylamide gel electrophoresis (PAGE) analysis, samples were heated at 90 °C for 5 min in an elution buffer, and electrophoresed in 1 mm-thick 12.5% SDS-polyacrylamide gels (Mini Protean II System, Bio-Rad), which were silver-stained as previously described [ ].

    Techniques: Staining, SDS Page, Affinity Chromatography, Molecular Weight, Marker

    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Blotting Analysis of HCVcp-Transient Expression in Tobacco Leaves A). Dot blotting; at control column: row 1: negative control (25 μg TSP of untransformed tobacco leaves); row 2: 5 μg positive control (eHCVcp). At PVX column: negative control (tobacco leaves transformed by PVX vector alone; ie, without Tr-HCVcp). PVX-core and pBI-core columns: the extracts from the PVX-core and pBI-core P19 co-agroinfiltrated leaves, respectively. Rows 2 and 1 in these two last columns correspond to 5 µg and 25 µg of TSP, respectively. B) Coomassie-stained 12% SDS-PAGE gel, loaded with; lane 1: 5 μg concentrated plant-purified HCVcp (from the PVX-core expression system). Lane 2: 5 μg of purified eHCVcp. Lane 3: crude protein extract from agroinfiltrated leaves with PVX-core (20 µg). Lane 4: untransformed leaves (20 µg). C) Western blotting; lane 1: positive control (700 ng purified eHCVcp). Lane 2: purified pHCVcp (700 ng from the PVX-core expression system). Lanes 3 and 4: the extracts from P19 co-agroinfiltrated PVX-core and pBI-core leaves, respectively (50 µg of plant TSP was applied in each lane). Lane 5: negative control (50 µg of plant TSP of untransformed tobacco leaves). Lane M: prestained protein ladder (Fermentas). HCVcp denote to HCV core protein, eHCVcp and pHCVcp dnote to E.coli -derived and plant-derived HCVcp, respectively. In western blot and SDS-PAGE figures, the location of HCVcp under the 20 kDa molecular weight range is shown by arrows. The reason for multiple bands in the case of eHCVcp is explained in the corresponding result section of the text. The results of SDS-PAGE showed that Ni-NTA pull-downs of plant extract contained endogenous nonspecific plant proteins besides pHCVcp. According to ELISA data, the concentration of pHCVcp was 1/20 of the column-purified protein. Therefore, although 1 µg (100 µL of 10 μg/mL coated) was coated, only a small amount reacted (less than 50 ng) ( Figure 5 B ). However, none of these endogenous nonspecific plant proteins reacted with anti-HCVcp in western blotting of the purified protein fraction.

    Journal: Hepatitis Monthly

    Article Title: Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications

    doi: 10.5812/hepatmon.20524

    Figure Lengend Snippet: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Blotting Analysis of HCVcp-Transient Expression in Tobacco Leaves A). Dot blotting; at control column: row 1: negative control (25 μg TSP of untransformed tobacco leaves); row 2: 5 μg positive control (eHCVcp). At PVX column: negative control (tobacco leaves transformed by PVX vector alone; ie, without Tr-HCVcp). PVX-core and pBI-core columns: the extracts from the PVX-core and pBI-core P19 co-agroinfiltrated leaves, respectively. Rows 2 and 1 in these two last columns correspond to 5 µg and 25 µg of TSP, respectively. B) Coomassie-stained 12% SDS-PAGE gel, loaded with; lane 1: 5 μg concentrated plant-purified HCVcp (from the PVX-core expression system). Lane 2: 5 μg of purified eHCVcp. Lane 3: crude protein extract from agroinfiltrated leaves with PVX-core (20 µg). Lane 4: untransformed leaves (20 µg). C) Western blotting; lane 1: positive control (700 ng purified eHCVcp). Lane 2: purified pHCVcp (700 ng from the PVX-core expression system). Lanes 3 and 4: the extracts from P19 co-agroinfiltrated PVX-core and pBI-core leaves, respectively (50 µg of plant TSP was applied in each lane). Lane 5: negative control (50 µg of plant TSP of untransformed tobacco leaves). Lane M: prestained protein ladder (Fermentas). HCVcp denote to HCV core protein, eHCVcp and pHCVcp dnote to E.coli -derived and plant-derived HCVcp, respectively. In western blot and SDS-PAGE figures, the location of HCVcp under the 20 kDa molecular weight range is shown by arrows. The reason for multiple bands in the case of eHCVcp is explained in the corresponding result section of the text. The results of SDS-PAGE showed that Ni-NTA pull-downs of plant extract contained endogenous nonspecific plant proteins besides pHCVcp. According to ELISA data, the concentration of pHCVcp was 1/20 of the column-purified protein. Therefore, although 1 µg (100 µL of 10 μg/mL coated) was coated, only a small amount reacted (less than 50 ng) ( Figure 5 B ). However, none of these endogenous nonspecific plant proteins reacted with anti-HCVcp in western blotting of the purified protein fraction.

    Article Snippet: For SDS-PAGE and western blotting analyses, purified HCV core from E. coli and plant and plant crude extracts of the recombinant HCV core protein expressed by P19 co-agroinfiltrated pBI121 and PVX vectors were loaded onto a 12% SDS-polyacrylamide gel; by the end of electrophoresis, the protein bands were either stained with coomassie brilliant blue (Bio-Rad) or transferred to the PVDF membrane.

    Techniques: Polyacrylamide Gel Electrophoresis, Expressing, Negative Control, Positive Control, Transformation Assay, Plasmid Preparation, Staining, SDS Page, Purification, Western Blot, Derivative Assay, Molecular Weight, Enzyme-linked Immunosorbent Assay, Concentration Assay

    ) and separated by SDS/PAGE. The glycoproteins and receptors indicated to the Left were identified by WB. For each sample, three replicate gels were developed as follows: one for gB and gD, one for gH and gL, and a third one for αv-integrin and nectin1. ( A ) The complex pulled down by αv STREP β6-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6-integrin lacks gL (lane 1, fuchsia star). In contrast, the complex assembled onto αvβ6-integrin alone (lane 4, green star) or nectin1 alone (lane 2) contains gL. ( B ) The complex assembled on αvβ8-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β8-integrin lacks gL (lane 8, fuchsia star). ( C ) The complex assembled on αvβ6N1-chimera in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6N-chimera lacks gL (lane 11, fuchsia star). ( D – F ) WB analysis of lysates from samples A – C ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A – C .

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

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: ) and separated by SDS/PAGE. The glycoproteins and receptors indicated to the Left were identified by WB. For each sample, three replicate gels were developed as follows: one for gB and gD, one for gH and gL, and a third one for αv-integrin and nectin1. ( A ) The complex pulled down by αv STREP β6-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6-integrin lacks gL (lane 1, fuchsia star). In contrast, the complex assembled onto αvβ6-integrin alone (lane 4, green star) or nectin1 alone (lane 2) contains gL. ( B ) The complex assembled on αvβ8-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β8-integrin lacks gL (lane 8, fuchsia star). ( C ) The complex assembled on αvβ6N1-chimera in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6N-chimera lacks gL (lane 11, fuchsia star). ( D – F ) WB analysis of lysates from samples A – C ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A – C .

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: SDS Page, Western Blot, Transfection

    , and separated by SDS/PAGE. Glycoproteins and receptors were identified by WB. It can be seen that the complex assembled onto αvβ6-integrin in the presence of nectin1 lacks gL (lane 3, fuchsia star). When gD (lane 4, HLB), gB (lane 8, DHL), or nectin1 (lanes 5, 6, 9, and αβ6) were missing, the complexes contained gL. ( C and D ) WB analysis of lysates from samples A and B ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A and B .

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

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: , and separated by SDS/PAGE. Glycoproteins and receptors were identified by WB. It can be seen that the complex assembled onto αvβ6-integrin in the presence of nectin1 lacks gL (lane 3, fuchsia star). When gD (lane 4, HLB), gB (lane 8, DHL), or nectin1 (lanes 5, 6, 9, and αβ6) were missing, the complexes contained gL. ( C and D ) WB analysis of lysates from samples A and B ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A and B .

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: SDS Page, Western Blot

    Block of HSV-1 infection by the neutralizing MAb LP11 to gH prevents the dissociation of gL from virion gH/gL. HSV-1(F) virions were preincubated with MAbs 52S, 53S, LP11, or IgGs. J cells expressing αvβ6-integrin plus nectin1 (N+αβ6), or nectin1 alone (N) were exposed to the preincubated virions. After 30 min, the media were harvested and subjected to SDS/PAGE and WB for gL. The medium of cells exposed to LP11-treated virions lacks gL. Lane HSV to the Left shows the WB reactivity of the indicated virion glycoproteins.

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

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: Block of HSV-1 infection by the neutralizing MAb LP11 to gH prevents the dissociation of gL from virion gH/gL. HSV-1(F) virions were preincubated with MAbs 52S, 53S, LP11, or IgGs. J cells expressing αvβ6-integrin plus nectin1 (N+αβ6), or nectin1 alone (N) were exposed to the preincubated virions. After 30 min, the media were harvested and subjected to SDS/PAGE and WB for gL. The medium of cells exposed to LP11-treated virions lacks gL. Lane HSV to the Left shows the WB reactivity of the indicated virion glycoproteins.

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: Blocking Assay, Infection, Expressing, SDS Page, Western Blot

    SDS-PAGE and Western blot of extracts from parts of the body of adults H. armigera and H. assulta . Upper panels: SDS-PAGE; lower panels: Western blot. (A); antennae, (P): proboscis, (T): tarsi, (W): wings of males (m) and females (f). The expression of OBP7 is limited to antennae with no significant differences between sexes or species. A weak staining in the extract of tarsi might indicate low levels of expression of OBP7 in such organ or cross-reactivity with other OBPs. Molecular weight markers ( M ) are as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Lysine at the C-Terminus of an Odorant-Binding Protein is Involved in Binding Aldehyde Pheromone Components in Two Helicoverpa Species

    doi: 10.1371/journal.pone.0055132

    Figure Lengend Snippet: SDS-PAGE and Western blot of extracts from parts of the body of adults H. armigera and H. assulta . Upper panels: SDS-PAGE; lower panels: Western blot. (A); antennae, (P): proboscis, (T): tarsi, (W): wings of males (m) and females (f). The expression of OBP7 is limited to antennae with no significant differences between sexes or species. A weak staining in the extract of tarsi might indicate low levels of expression of OBP7 in such organ or cross-reactivity with other OBPs. Molecular weight markers ( M ) are as in Figure 1 .

    Article Snippet: Western Blot Analysis After electrophoretic separation under 14% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), duplicate gels were stained with 0.1% Coomassie blue R250 in 10% acetic acid, 20% ethanol or electroblotted on Trans-Blot nitrocellulose membrane (Bio-Rad Lab) by the procedure of Kyhse-Andersen .

    Techniques: SDS Page, Western Blot, Expressing, Staining, Molecular Weight

    Kap60 and Yar1 compete for Rps3 binding. ( a ) Kap60 co-elutes after Rps3-TAP purification. An RPS3 -TAP strain carrying a KAP60 -3xHA fusion was subjected to TAP purification. A KAP60 -3xHA strain without TAP-tag served as negative control (untagged). Lysates and final eluates (E-TAP) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. Note that there is some unspecific recovery of Kap60-3xHA in the eluate of the control strain; however, the protein is clearly enriched in the Rps3-TAP eluate. Kap123: Kap123 was detected in this band by mass spectrometry (Mascot score 211, 11 matched peptides, 12% sequence coverage). Kap60-3xHA: Kap60 was detected in this band by mass spectrometry (Mascot score 85, 4 matched peptides, 10% sequence coverage). ( b ) GST-tagged importins (Kap60ΔIBB or Kap123) were immobilized on glutathione-agarose beads. Truncated Kap60 lacking the N-terminal IBB domain (80 amino acids) was used in order to prevent inhibition of cargo binding by this domain in the absence of Kap95. Beads were incubated with purified, recombinant His6-Rps3/Flag-Yar1 complex or purified Flag-Yar1. Importins and bound material were eluted by boiling. Eluates were analyzed by SDS-PAGE and Coomassie staining or Western blotting with indicated antibodies. Asterisks in the Coomassie stained gel mark the respective importins, circles indicate bound Rps3. ( c ) Disruption of the Rps3-NLS impairs Kap60 binding. GST-tagged Kap60ΔIBB was incubated with His6-Rps3/Flag-Yar1 complex containing wild-type Rps3 or Rps3(K7/K10 > A) mutant protein. Kap60ΔIBB was eluted by TEV-cleavage. Eluates were analyzed by SDS-PAGE and Coomassie staining or Western blotting.

    Journal: Scientific Reports

    Article Title: Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

    doi: 10.1038/srep36714

    Figure Lengend Snippet: Kap60 and Yar1 compete for Rps3 binding. ( a ) Kap60 co-elutes after Rps3-TAP purification. An RPS3 -TAP strain carrying a KAP60 -3xHA fusion was subjected to TAP purification. A KAP60 -3xHA strain without TAP-tag served as negative control (untagged). Lysates and final eluates (E-TAP) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. Note that there is some unspecific recovery of Kap60-3xHA in the eluate of the control strain; however, the protein is clearly enriched in the Rps3-TAP eluate. Kap123: Kap123 was detected in this band by mass spectrometry (Mascot score 211, 11 matched peptides, 12% sequence coverage). Kap60-3xHA: Kap60 was detected in this band by mass spectrometry (Mascot score 85, 4 matched peptides, 10% sequence coverage). ( b ) GST-tagged importins (Kap60ΔIBB or Kap123) were immobilized on glutathione-agarose beads. Truncated Kap60 lacking the N-terminal IBB domain (80 amino acids) was used in order to prevent inhibition of cargo binding by this domain in the absence of Kap95. Beads were incubated with purified, recombinant His6-Rps3/Flag-Yar1 complex or purified Flag-Yar1. Importins and bound material were eluted by boiling. Eluates were analyzed by SDS-PAGE and Coomassie staining or Western blotting with indicated antibodies. Asterisks in the Coomassie stained gel mark the respective importins, circles indicate bound Rps3. ( c ) Disruption of the Rps3-NLS impairs Kap60 binding. GST-tagged Kap60ΔIBB was incubated with His6-Rps3/Flag-Yar1 complex containing wild-type Rps3 or Rps3(K7/K10 > A) mutant protein. Kap60ΔIBB was eluted by TEV-cleavage. Eluates were analyzed by SDS-PAGE and Coomassie staining or Western blotting.

    Article Snippet: Eluates were analyzed on 12% or 4–12% SDS-polyacrylamide gels (Invitrogen) and Coomassie staining or Western blotting.

    Techniques: Binding Assay, Purification, Negative Control, SDS Page, Staining, Western Blot, Mass Spectrometry, Sequencing, Inhibition, Incubation, Recombinant, Mutagenesis

    Yar1 and Kap60 are genetically and physically linked. (a) Growth defect of a yar1 deletion strain is enhanced in combination with kap60- ts and kap95- ts mutant alleles. kap60- ts/ yar1 Δ or kap95- ts/ yar1 Δ strains were transformed with plasmids harboring the indicated wild-type alleles or empty plasmids (−). Cells were spotted in 10-fold serial dilutions on SD-Leu-Trp plates and incubated at the indicated temperatures. ( b ) Kap60 is co-purified with Yar1-TAP. A YAR1- TAP strain carrying a KAP60 -3xHA fusion was subjected to TAP purification. A strain containing KAP60 -3xHA but no TAP-tag served as negative control (untagged). Lysates and final eluates (E-TAP) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. Kap60-3xHA: Kap60 was detected in this band by mass spectrometry (Mascot score 255, 5 matched peptides, 15% sequence coverage).

    Journal: Scientific Reports

    Article Title: Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

    doi: 10.1038/srep36714

    Figure Lengend Snippet: Yar1 and Kap60 are genetically and physically linked. (a) Growth defect of a yar1 deletion strain is enhanced in combination with kap60- ts and kap95- ts mutant alleles. kap60- ts/ yar1 Δ or kap95- ts/ yar1 Δ strains were transformed with plasmids harboring the indicated wild-type alleles or empty plasmids (−). Cells were spotted in 10-fold serial dilutions on SD-Leu-Trp plates and incubated at the indicated temperatures. ( b ) Kap60 is co-purified with Yar1-TAP. A YAR1- TAP strain carrying a KAP60 -3xHA fusion was subjected to TAP purification. A strain containing KAP60 -3xHA but no TAP-tag served as negative control (untagged). Lysates and final eluates (E-TAP) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. Kap60-3xHA: Kap60 was detected in this band by mass spectrometry (Mascot score 255, 5 matched peptides, 15% sequence coverage).

    Article Snippet: Eluates were analyzed on 12% or 4–12% SDS-polyacrylamide gels (Invitrogen) and Coomassie staining or Western blotting.

    Techniques: Mutagenesis, Transformation Assay, Incubation, Purification, Negative Control, SDS Page, Staining, Western Blot, Mass Spectrometry, Sequencing

    Kap60/Kap95 orchestrate nuclear import of the Rps3/Rps3/Yar1 complex. ( a ) Kap60/Kap95 are bound to the Rps3/Yar1 complex in vivo . The cell lysate of a YAR1 -TAP, RPS3 -Flag strain was subjected to affinity purification via the protein A tag of Yar1. The obtained TEV (tobacco etch virus) eluate was further purified in a second affinity purification step via Rps3-Flag. As negative control the lysate of an untagged wild-type strain was subjected to the same purification procedure. Lysates and final eluates (E-Flag) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. ( b ) Kap60 binds the Rps3/Yar1 complex in vitro . His6-Rps3/Flag-Yar1 complex was immobilized via His6-Rps3 on Ni-NTA-agarose beads and incubated with increasing amounts of purified Kap60ΔIBB. After elution of His6-Rps3 (Ni-NTA-eluate), bound material was further subjected to Flag-purification via Flag-Yar1 (Flag-eluate). As negative control, empty Ni-NTA agarose or Flag-agarose beads were incubated with the highest Kap60ΔIBB concentration. The eluates were analyzed by SDS-PAGE and Coomassie staining. ( c ) Model describing the formation of the Yar1/Rps3/Rps3/Kap60/Kap95 import-complex that allows simultaneous targeting of an Rps3 dimer bound to one Yar1 chaperone into the nucleus.

    Journal: Scientific Reports

    Article Title: Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

    doi: 10.1038/srep36714

    Figure Lengend Snippet: Kap60/Kap95 orchestrate nuclear import of the Rps3/Rps3/Yar1 complex. ( a ) Kap60/Kap95 are bound to the Rps3/Yar1 complex in vivo . The cell lysate of a YAR1 -TAP, RPS3 -Flag strain was subjected to affinity purification via the protein A tag of Yar1. The obtained TEV (tobacco etch virus) eluate was further purified in a second affinity purification step via Rps3-Flag. As negative control the lysate of an untagged wild-type strain was subjected to the same purification procedure. Lysates and final eluates (E-Flag) were analyzed by SDS-PAGE followed by Coomassie staining or Western blotting with the indicated antibodies. ( b ) Kap60 binds the Rps3/Yar1 complex in vitro . His6-Rps3/Flag-Yar1 complex was immobilized via His6-Rps3 on Ni-NTA-agarose beads and incubated with increasing amounts of purified Kap60ΔIBB. After elution of His6-Rps3 (Ni-NTA-eluate), bound material was further subjected to Flag-purification via Flag-Yar1 (Flag-eluate). As negative control, empty Ni-NTA agarose or Flag-agarose beads were incubated with the highest Kap60ΔIBB concentration. The eluates were analyzed by SDS-PAGE and Coomassie staining. ( c ) Model describing the formation of the Yar1/Rps3/Rps3/Kap60/Kap95 import-complex that allows simultaneous targeting of an Rps3 dimer bound to one Yar1 chaperone into the nucleus.

    Article Snippet: Eluates were analyzed on 12% or 4–12% SDS-polyacrylamide gels (Invitrogen) and Coomassie staining or Western blotting.

    Techniques: In Vivo, Affinity Purification, Purification, Negative Control, SDS Page, Staining, Western Blot, In Vitro, Incubation, Concentration Assay

    Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% SDS-PAGE gels and transferred to PVDF membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments

    Journal: The Journal of steroid biochemistry and molecular biology

    Article Title: Functional characterization of the G162R and D216H genetic variants of human CYP17A1

    doi: 10.1016/j.jsbmb.2017.12.002

    Figure Lengend Snippet: Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% SDS-PAGE gels and transferred to PVDF membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments

    Article Snippet: Protein (30–50 μg per lane) was loaded, resolved on a 10% SDS-polyacrylamide gel (ThermoScientific), and transferred onto a PVDF membrane.

    Techniques: Variant Assay, Transfection, Construct, Lysis, Protease Inhibitor, SDS Page

    SDS-PAGE of the Proteus mirabilis strain ATCC 29245. The purified cytochrome b was loaded on 12.5% gel. The gel was stained with commassie brilliant blue after electrophoresis. Lane 1 (molecular mass marker proteins), lane 2 (sample 1, partially purified complex II after ion exchange chromatography) and lane 3, (sample 2, purified complex II). The molecular masses of three subunits of the Proteus mirabilis strain ATCC 29245 complex II were estimated to be 68, 28.5, and 19.5 kDa.

    Journal: Brazilian Journal of Microbiology

    Article Title: Isolation and Purification of Complex II from Proteus Mirabilis Strain ATCC 29245

    doi: 10.1590/S1517-83822010005000032

    Figure Lengend Snippet: SDS-PAGE of the Proteus mirabilis strain ATCC 29245. The purified cytochrome b was loaded on 12.5% gel. The gel was stained with commassie brilliant blue after electrophoresis. Lane 1 (molecular mass marker proteins), lane 2 (sample 1, partially purified complex II after ion exchange chromatography) and lane 3, (sample 2, purified complex II). The molecular masses of three subunits of the Proteus mirabilis strain ATCC 29245 complex II were estimated to be 68, 28.5, and 19.5 kDa.

    Article Snippet: In the estimation of the apparent molecular weight by SDS-polyacrylamide gel electrophoresis, a set of protein markers (PAGE ruler pre-stained protein ladder, SM0671 Fermentas) with known molecular weights were used.

    Techniques: SDS Page, Purification, Staining, Electrophoresis, Marker, Ion Exchange Chromatography

    In vitro characterization of SecE(S105P) effects on pro-OmpA translocation. (A) Effects on different SecY mutational defects. IMVs were prepared from strains HM808 ( secY39 secE105 ) (lanes 1 and 2), GN04 ( secY39 ) (lanes 3 and 4), HM809 ( secY205 secE105 ) (lanes 5 and 6), GN05 ( secY205 ) (lanes 7 and 8), HM810 ( secY104 secE105 ) (lanes 9 and 10), GN09 ( secY104 ) (lanes 11 and 12), and TW156 ( secY + secE + ) (lanes 13 and 14). They were subjected to an in vitro translocation assay using 35 S-labeled pro-OmpA and wild-type SecA. The PMF was generated (+) or dissipated (−) by addition of 5 mM succinate or 10 μM carbonycyanide- m -chlorophenyl hydrazone, respectively. Reactions were allowed to proceed for 10 min at 37°C (upper panel) or 20°C (lower panel). Samples were treated with proteinase K and analyzed by SDS-PAGE and phosphorimager exposure. p, precursor forms; m, mature forms. (B) pro-OmpA translocation reaction mediated by the SecE(S105P) single-mutant IMV. IMVs were prepared from strains HM811 ( secE105 ) (lanes 1 to 10) and TW156 ( secE + ) (lanes 11 to 20). They were subjected to a 35 S-labeled pro-OmpA translocation assay at 20°C in the presence (+) or absence (−) of the PMF for the indicated lengths of time. Shown are SDS-PAGEprofiles after proteinase K treatment. i, fragments generated from incompletely translocated pro-OmpA. (C) Time courses of full-length translocation. Intensities of the full-length proOmpA product (mature and precursor forms) with a SecE(S105P) IMV (circles) or a Sec + IMV (triangles) are plotted against the reaction time. Open and solid symbols, results in the presence and absence of the PMF, respectively. (D) Time courses of generation of incompletely translocated products. Intensities of the incomplete translocation products (corresponding to the “i” bands in panel B) are plotted against the reaction time. Symbols are the same as in panel C.

    Journal: Journal of Bacteriology

    Article Title: A SecE Mutation That Modulates SecY-SecE Translocase Assembly, Identified as a Specific Suppressor of SecY Defects

    doi: 10.1128/JB.185.3.948-956.2003

    Figure Lengend Snippet: In vitro characterization of SecE(S105P) effects on pro-OmpA translocation. (A) Effects on different SecY mutational defects. IMVs were prepared from strains HM808 ( secY39 secE105 ) (lanes 1 and 2), GN04 ( secY39 ) (lanes 3 and 4), HM809 ( secY205 secE105 ) (lanes 5 and 6), GN05 ( secY205 ) (lanes 7 and 8), HM810 ( secY104 secE105 ) (lanes 9 and 10), GN09 ( secY104 ) (lanes 11 and 12), and TW156 ( secY + secE + ) (lanes 13 and 14). They were subjected to an in vitro translocation assay using 35 S-labeled pro-OmpA and wild-type SecA. The PMF was generated (+) or dissipated (−) by addition of 5 mM succinate or 10 μM carbonycyanide- m -chlorophenyl hydrazone, respectively. Reactions were allowed to proceed for 10 min at 37°C (upper panel) or 20°C (lower panel). Samples were treated with proteinase K and analyzed by SDS-PAGE and phosphorimager exposure. p, precursor forms; m, mature forms. (B) pro-OmpA translocation reaction mediated by the SecE(S105P) single-mutant IMV. IMVs were prepared from strains HM811 ( secE105 ) (lanes 1 to 10) and TW156 ( secE + ) (lanes 11 to 20). They were subjected to a 35 S-labeled pro-OmpA translocation assay at 20°C in the presence (+) or absence (−) of the PMF for the indicated lengths of time. Shown are SDS-PAGEprofiles after proteinase K treatment. i, fragments generated from incompletely translocated pro-OmpA. (C) Time courses of full-length translocation. Intensities of the full-length proOmpA product (mature and precursor forms) with a SecE(S105P) IMV (circles) or a Sec + IMV (triangles) are plotted against the reaction time. Open and solid symbols, results in the presence and absence of the PMF, respectively. (D) Time courses of generation of incompletely translocated products. Intensities of the incomplete translocation products (corresponding to the “i” bands in panel B) are plotted against the reaction time. Symbols are the same as in panel C.

    Article Snippet: Then the samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) ( ) and immunoblotting with anti-SecY , anti-SecE (which was raised against a synthetic peptide with a sequence corresponding to residues 1 to 16 of SecE), anti-SecG , and anti-Myc (purchased from Santa Cruz Biotechnology) antibodies.

    Techniques: In Vitro, Translocation Assay, Labeling, Peptide Mass Fingerprinting, Generated, SDS Page, Mutagenesis, Size-exclusion Chromatography

    SecY-stabilizing ability of SecE(S105P). Strain HM834 ( secE + ) or HM835 ( secE105 ) was transformed with two compatible plasmids, one overexpressing SecY or its derivative and another overexpressing SecE + (lower panel) or SecE(S105P) (upper panel), respectively. The SecY plasmids used were pCM10 (SecY + ) (lanes 1 to 5), pHM404 [SecY Δ(346-357)] (lanes 6 to 10), and pHM405 [SecY(R357E)] (lanes 11 to 15). Cells were grown in the presence of 1 mM IPTG and 5 mM cyclic AMP at 37°C and were pulse-labeled with [ 35 S]methionine for 30 s, followed by a chase with unlabeled methionine for the indicated periods. The labeled SecY proteins were immunoprecipitated and visualized by phosphor image analyzer after SDS-PAGE.

    Journal: Journal of Bacteriology

    Article Title: A SecE Mutation That Modulates SecY-SecE Translocase Assembly, Identified as a Specific Suppressor of SecY Defects

    doi: 10.1128/JB.185.3.948-956.2003

    Figure Lengend Snippet: SecY-stabilizing ability of SecE(S105P). Strain HM834 ( secE + ) or HM835 ( secE105 ) was transformed with two compatible plasmids, one overexpressing SecY or its derivative and another overexpressing SecE + (lower panel) or SecE(S105P) (upper panel), respectively. The SecY plasmids used were pCM10 (SecY + ) (lanes 1 to 5), pHM404 [SecY Δ(346-357)] (lanes 6 to 10), and pHM405 [SecY(R357E)] (lanes 11 to 15). Cells were grown in the presence of 1 mM IPTG and 5 mM cyclic AMP at 37°C and were pulse-labeled with [ 35 S]methionine for 30 s, followed by a chase with unlabeled methionine for the indicated periods. The labeled SecY proteins were immunoprecipitated and visualized by phosphor image analyzer after SDS-PAGE.

    Article Snippet: Then the samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) ( ) and immunoblotting with anti-SecY , anti-SecE (which was raised against a synthetic peptide with a sequence corresponding to residues 1 to 16 of SecE), anti-SecG , and anti-Myc (purchased from Santa Cruz Biotechnology) antibodies.

    Techniques: Transformation Assay, Labeling, Immunoprecipitation, SDS Page

    Stability of the SecY-SecE association in DM. Two compatible plasmids, pSTD343 ( lacI q ) and pNA3 ( secY- His 6 -myc ), were introduced into strains HM562 ( secE + ) and HM564 ( secE105 ). Membrane fractions were prepared from lac -induced cultures of the plasmid-bearing cells, and portions containing 100 μg of protein were solubilized with 2% DM. Supernatants after centrifugation (at 100,000 × g for 30 min at 4°C) were incubated at the indicated temperature for 30 min and then applied to Ni-NTA spin columns (Qiagen), which were washed with a buffer containing 0.03% DM and eluted with 1 M imidazole in the same buffer. Eluates were subjected to SDS-PAGE and immunoblotting with the antibodies shown on the left.

    Journal: Journal of Bacteriology

    Article Title: A SecE Mutation That Modulates SecY-SecE Translocase Assembly, Identified as a Specific Suppressor of SecY Defects

    doi: 10.1128/JB.185.3.948-956.2003

    Figure Lengend Snippet: Stability of the SecY-SecE association in DM. Two compatible plasmids, pSTD343 ( lacI q ) and pNA3 ( secY- His 6 -myc ), were introduced into strains HM562 ( secE + ) and HM564 ( secE105 ). Membrane fractions were prepared from lac -induced cultures of the plasmid-bearing cells, and portions containing 100 μg of protein were solubilized with 2% DM. Supernatants after centrifugation (at 100,000 × g for 30 min at 4°C) were incubated at the indicated temperature for 30 min and then applied to Ni-NTA spin columns (Qiagen), which were washed with a buffer containing 0.03% DM and eluted with 1 M imidazole in the same buffer. Eluates were subjected to SDS-PAGE and immunoblotting with the antibodies shown on the left.

    Article Snippet: Then the samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) ( ) and immunoblotting with anti-SecY , anti-SecE (which was raised against a synthetic peptide with a sequence corresponding to residues 1 to 16 of SecE), anti-SecG , and anti-Myc (purchased from Santa Cruz Biotechnology) antibodies.

    Techniques: Plasmid Preparation, Centrifugation, Incubation, SDS Page

    SecE(S105P) preferentially compensates for the early translocation defect of the SecY39 alteration. (A) Translocation. IMVs prepared from strains GN04 ( secY39 ) (lanes 1 to 5), HM808 ( secY39 secE105 ) (lanes 6 to 10), and TW156 ( secY + ) (lanes 11 to 15) were subjected to a 35 S-labeled pro-OmpA translocation reaction at 37°C with wild type SecA and in the absence of the PMF. At the indicated time points, samples were treated with proteinase K and analyzed by SDS-PAGE and phosphorimaging. Positions of precursor (p) and mature (m) forms, as well as those of two intermediate forms (I 26 and I 16 ), are shown. (B) Processing. The reactions for which results are shown in panel A were terminated directly with 5% TCA. Precursor and mature forms of OmpA were separated by SDS-PAGE. (C) Quantitative representations of data from panels A and B. Inten-sities of full-length translocation products (circles) and the intermediates I 26 (triangles) and I 16 (squares) in panel A are plotted against reaction time. Processing efficiencies from panel B are also shown (solid diamonds). Left and right graphs show results with the SecY39-SecE(S105P) IMV and the Sec + IMV, respectively. (D) Further translocation of I 16 by a superactive SecA derivative. IMVs from HM808 ( secY39 secE105 ) were subjected to a 35 S-labeled pro-OmpA translocation reaction at 37°C with wild-type SecA (final concentration, 10 μg/ml) in the absence of the PMF. After 5 min, either SecA329 or wild-type SecA (final concentration, 20 μg/ml) was added to the reaction mixture, followed by further incubation at 37°C. At the indicated time points, samples were treated with proteinase K followed by SDS-PAGE and phosphorimaging. (E) Quantitative representations of the data from panel D. Symbols are the same as in panel C.

    Journal: Journal of Bacteriology

    Article Title: A SecE Mutation That Modulates SecY-SecE Translocase Assembly, Identified as a Specific Suppressor of SecY Defects

    doi: 10.1128/JB.185.3.948-956.2003

    Figure Lengend Snippet: SecE(S105P) preferentially compensates for the early translocation defect of the SecY39 alteration. (A) Translocation. IMVs prepared from strains GN04 ( secY39 ) (lanes 1 to 5), HM808 ( secY39 secE105 ) (lanes 6 to 10), and TW156 ( secY + ) (lanes 11 to 15) were subjected to a 35 S-labeled pro-OmpA translocation reaction at 37°C with wild type SecA and in the absence of the PMF. At the indicated time points, samples were treated with proteinase K and analyzed by SDS-PAGE and phosphorimaging. Positions of precursor (p) and mature (m) forms, as well as those of two intermediate forms (I 26 and I 16 ), are shown. (B) Processing. The reactions for which results are shown in panel A were terminated directly with 5% TCA. Precursor and mature forms of OmpA were separated by SDS-PAGE. (C) Quantitative representations of data from panels A and B. Inten-sities of full-length translocation products (circles) and the intermediates I 26 (triangles) and I 16 (squares) in panel A are plotted against reaction time. Processing efficiencies from panel B are also shown (solid diamonds). Left and right graphs show results with the SecY39-SecE(S105P) IMV and the Sec + IMV, respectively. (D) Further translocation of I 16 by a superactive SecA derivative. IMVs from HM808 ( secY39 secE105 ) were subjected to a 35 S-labeled pro-OmpA translocation reaction at 37°C with wild-type SecA (final concentration, 10 μg/ml) in the absence of the PMF. After 5 min, either SecA329 or wild-type SecA (final concentration, 20 μg/ml) was added to the reaction mixture, followed by further incubation at 37°C. At the indicated time points, samples were treated with proteinase K followed by SDS-PAGE and phosphorimaging. (E) Quantitative representations of the data from panel D. Symbols are the same as in panel C.

    Article Snippet: Then the samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) ( ) and immunoblotting with anti-SecY , anti-SecE (which was raised against a synthetic peptide with a sequence corresponding to residues 1 to 16 of SecE), anti-SecG , and anti-Myc (purchased from Santa Cruz Biotechnology) antibodies.

    Techniques: Translocation Assay, Labeling, Peptide Mass Fingerprinting, SDS Page, Size-exclusion Chromatography, Concentration Assay, Incubation

    Trx and TrxR expression and localization. (A) The levels of Trx and TrxR were examined in nuclear extracts (10 µg) from human ERα-positive breast (MCF-7) and ERα-negative breast (MDA-MB-231), U2 osteosarcoma (U2OS), and cervical (HeLa) cancer cells. (B) Trx and TrxR expression was examined in MCF-7 cells that had been treated with ethanol (−E 2 ) or 10 nM E 2 (+E 2 ) for 24 h. Samples were fractionated by SDS-PAGE and subjected to western analysis with a Trx- or TrxR-specific antibody. GAPDH levels were monitored to demonstrate that similar amounts of sample were loaded in each lane. (C) MCF-7 cells were treated with ethanol (−E 2 ) or 10 nM E 2 (+E 2 ) for 24 h. Expression of Trx and TrxR was monitored using immunocyto-chemistry with Trx- and TrxR-specific antibodies. DAPI counter-staining was used to detect cell nuclei. The insert in the upper right hand corner of the −E 2 .

    Journal: Journal of molecular endocrinology

    Article Title: Thioredoxin and thioredoxin reductase influence estrogen receptor ?-mediated gene expression in human breast cancer cells

    doi: 10.1677/JME-09-0053

    Figure Lengend Snippet: Trx and TrxR expression and localization. (A) The levels of Trx and TrxR were examined in nuclear extracts (10 µg) from human ERα-positive breast (MCF-7) and ERα-negative breast (MDA-MB-231), U2 osteosarcoma (U2OS), and cervical (HeLa) cancer cells. (B) Trx and TrxR expression was examined in MCF-7 cells that had been treated with ethanol (−E 2 ) or 10 nM E 2 (+E 2 ) for 24 h. Samples were fractionated by SDS-PAGE and subjected to western analysis with a Trx- or TrxR-specific antibody. GAPDH levels were monitored to demonstrate that similar amounts of sample were loaded in each lane. (C) MCF-7 cells were treated with ethanol (−E 2 ) or 10 nM E 2 (+E 2 ) for 24 h. Expression of Trx and TrxR was monitored using immunocyto-chemistry with Trx- and TrxR-specific antibodies. DAPI counter-staining was used to detect cell nuclei. The insert in the upper right hand corner of the −E 2 .

    Article Snippet: Samples were washed thrice with 20 mM Tris pH 7.4, 10 mM EDTA, 100 mM NaCl, 0.1% NP-40, 1 mM Na3 VO4 , 50 mM NaF, and 1× PIC before fractionation on SDS-polyacrylamide gels and western analysis with an ERα-specific antibody (sc-543, Santa Cruz Biotechnologies).

    Techniques: Expressing, Multiple Displacement Amplification, SDS Page, Western Blot, Staining

    POLA1 levels are elevated in colorectal cancer and ST1926 decreases its expression. A. ST1926 reduces POLA1 protein levels in colorectal cell lines. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends in protein levels were observed in two independent experiments. B. POLA1 basal protein levels are elevated in CRC cell lines compared to the normal-like NCM460 cells. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends were observed in two independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. Expression levels of POLA1 ]. D. POLA1 ]. P values

    Journal: American Journal of Cancer Research

    Article Title: Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

    doi:

    Figure Lengend Snippet: POLA1 levels are elevated in colorectal cancer and ST1926 decreases its expression. A. ST1926 reduces POLA1 protein levels in colorectal cell lines. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends in protein levels were observed in two independent experiments. B. POLA1 basal protein levels are elevated in CRC cell lines compared to the normal-like NCM460 cells. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends were observed in two independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. Expression levels of POLA1 ]. D. POLA1 ]. P values

    Article Snippet: Total cellular proteins (50 µg) were loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis and immunoblotted with the following antibodies: p53 (sc-126), p21 (sc-397), and PARP (sc-7150) from Santa Cruz Biotechnology (Heidelberg, Germany), γ-H2AX (2577) from Cell Signaling, Danvers, MA, POLA1 (31777) from Abcam (Cambridge, UK), and GAPDH (MAB5476) (Abnova, Heidelberg, Germany).

    Techniques: Expressing

    ST1926-resistant cells are cross-resistant to CD437 and both compounds inhibit POLA1 activity. A. Effect of ST1926 treatment on the growth of human HCT116-STR. ST1926-resistant cells (HCT116-STR) were generated by treating the parental cell line with increasing concentrations of ST1926 over a period of eight months. HCT116-STR cells were seeded in 96-well plates at a density of 5,000 cells/well, and treated with the indicated concentrations of ST1926 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of three independent experiments ± SEM. B. HCT116-STR DNA damage response to ST1926 treatment. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to 24 hours. Total SDS protein lysates (50 µg/lane) were immunoblotted against γ-H2AX antibody. Similar trends in protein levels were observed in three independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. TUNEL analysis of HCT116-STR cells treated with 0.1% DMSO or 1 μM ST1926 up to 48 hours. Results are representative of two independent experiments. D. Effect of CD437 treatment on HCT116 and HCT116-STR cell growth. Cells were treated with the indicated concentrations of CD437 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of at least three independent experiments ± SEM. E. Primer extension assay was evaluated in the presence of increasing concentrations of ST1926 and CD437 (0.069, 0.21, 0.62, 1.9, 5.6, 17, 50, and 100 μM). “-” represents the control (DMSO). The activity of POLA1 was determined by the detection of primer of a 25-nucleotide product, and a gel representative of two independent experiments is shown.

    Journal: American Journal of Cancer Research

    Article Title: Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

    doi:

    Figure Lengend Snippet: ST1926-resistant cells are cross-resistant to CD437 and both compounds inhibit POLA1 activity. A. Effect of ST1926 treatment on the growth of human HCT116-STR. ST1926-resistant cells (HCT116-STR) were generated by treating the parental cell line with increasing concentrations of ST1926 over a period of eight months. HCT116-STR cells were seeded in 96-well plates at a density of 5,000 cells/well, and treated with the indicated concentrations of ST1926 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of three independent experiments ± SEM. B. HCT116-STR DNA damage response to ST1926 treatment. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to 24 hours. Total SDS protein lysates (50 µg/lane) were immunoblotted against γ-H2AX antibody. Similar trends in protein levels were observed in three independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. TUNEL analysis of HCT116-STR cells treated with 0.1% DMSO or 1 μM ST1926 up to 48 hours. Results are representative of two independent experiments. D. Effect of CD437 treatment on HCT116 and HCT116-STR cell growth. Cells were treated with the indicated concentrations of CD437 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of at least three independent experiments ± SEM. E. Primer extension assay was evaluated in the presence of increasing concentrations of ST1926 and CD437 (0.069, 0.21, 0.62, 1.9, 5.6, 17, 50, and 100 μM). “-” represents the control (DMSO). The activity of POLA1 was determined by the detection of primer of a 25-nucleotide product, and a gel representative of two independent experiments is shown.

    Article Snippet: Total cellular proteins (50 µg) were loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis and immunoblotted with the following antibodies: p53 (sc-126), p21 (sc-397), and PARP (sc-7150) from Santa Cruz Biotechnology (Heidelberg, Germany), γ-H2AX (2577) from Cell Signaling, Danvers, MA, POLA1 (31777) from Abcam (Cambridge, UK), and GAPDH (MAB5476) (Abnova, Heidelberg, Germany).

    Techniques: Activity Assay, Generated, MTT Assay, TUNEL Assay, Primer Extension Assay

    ST1926 treatment results in loss of mitochondrial membrane potential and early DNA damage, independently of p53 and p21. (A) Dissipation of the mitochondrial membrane potential of colorectal cancer cells by ST1926. HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells were treated with 0.1% DMSO or 1 µM ST1926 for two days, then stained with Rhodamine-123. Accumulation of Rhodamine-123 fluorescent dye was measured by flow cytometry, and is represented in histograms showing an overlay of ST1926-treated cells over control cells. Results are representative of three independent experiments and numbers indicate percentage of cells with mitochondrial dissipation. (B) ST1926 increases the protein levels of p53 and p21, and (C) γ-H2AX in HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total SDS protein lysates (50 µg/lane) were immunoblotted against p53, p21, and γ-H2AX antibodies. Similar trends in protein levels were observed in three independent experiments. All blots were reprobed with GAPDH antibody to ensure equal protein loading (B, C).

    Journal: American Journal of Cancer Research

    Article Title: Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

    doi:

    Figure Lengend Snippet: ST1926 treatment results in loss of mitochondrial membrane potential and early DNA damage, independently of p53 and p21. (A) Dissipation of the mitochondrial membrane potential of colorectal cancer cells by ST1926. HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells were treated with 0.1% DMSO or 1 µM ST1926 for two days, then stained with Rhodamine-123. Accumulation of Rhodamine-123 fluorescent dye was measured by flow cytometry, and is represented in histograms showing an overlay of ST1926-treated cells over control cells. Results are representative of three independent experiments and numbers indicate percentage of cells with mitochondrial dissipation. (B) ST1926 increases the protein levels of p53 and p21, and (C) γ-H2AX in HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total SDS protein lysates (50 µg/lane) were immunoblotted against p53, p21, and γ-H2AX antibodies. Similar trends in protein levels were observed in three independent experiments. All blots were reprobed with GAPDH antibody to ensure equal protein loading (B, C).

    Article Snippet: Total cellular proteins (50 µg) were loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis and immunoblotted with the following antibodies: p53 (sc-126), p21 (sc-397), and PARP (sc-7150) from Santa Cruz Biotechnology (Heidelberg, Germany), γ-H2AX (2577) from Cell Signaling, Danvers, MA, POLA1 (31777) from Abcam (Cambridge, UK), and GAPDH (MAB5476) (Abnova, Heidelberg, Germany).

    Techniques: Staining, Flow Cytometry, Cytometry

    ST1926 induces S-phase arrest and apoptosis in colorectal cancer cells. A. ST1926 treatment causes accumulation of cells in the sub-G 1 region. B. Cell cycle distribution of colorectal cancer cells. HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells were treated with 0.1% DMSO or 1 μM ST1926 up to two days and stained with propidium iodide (50 mg/ml). The sub-G 1 percentage presumably indicates apoptotic cells. The sum of G 0 /G 1 , S, and G 2 /M phases is a percentage of nonapoptotic cells at days one and two post-ST1926 treatment. Percentage cells in the G 0 /G 1 phase are calculated as 100 - (S+G 2 /M). Results represent the average of three independent experiments (± SEM). C. TUNEL analysis of colorectal cancer cells treated with 0.1% DMSO or 1 μM ST1926 up to two days. Histograms are representative of two independent experiments and numbers indicate percentage of TUNEL-positive cells. D. ST1926 causes PARP cleavage in colorectal cancer cells. Cells were treated with 0.1% DMSO or 1 μM ST1926 up to two days. Total SDS protein lysates (50 µg/lane) were immunoblotted against PARP antibody. Arrow indicates cleaved PARP subunit. Similar trends were observed in three independent experiments. *P

    Journal: American Journal of Cancer Research

    Article Title: Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

    doi:

    Figure Lengend Snippet: ST1926 induces S-phase arrest and apoptosis in colorectal cancer cells. A. ST1926 treatment causes accumulation of cells in the sub-G 1 region. B. Cell cycle distribution of colorectal cancer cells. HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells were treated with 0.1% DMSO or 1 μM ST1926 up to two days and stained with propidium iodide (50 mg/ml). The sub-G 1 percentage presumably indicates apoptotic cells. The sum of G 0 /G 1 , S, and G 2 /M phases is a percentage of nonapoptotic cells at days one and two post-ST1926 treatment. Percentage cells in the G 0 /G 1 phase are calculated as 100 - (S+G 2 /M). Results represent the average of three independent experiments (± SEM). C. TUNEL analysis of colorectal cancer cells treated with 0.1% DMSO or 1 μM ST1926 up to two days. Histograms are representative of two independent experiments and numbers indicate percentage of TUNEL-positive cells. D. ST1926 causes PARP cleavage in colorectal cancer cells. Cells were treated with 0.1% DMSO or 1 μM ST1926 up to two days. Total SDS protein lysates (50 µg/lane) were immunoblotted against PARP antibody. Arrow indicates cleaved PARP subunit. Similar trends were observed in three independent experiments. *P

    Article Snippet: Total cellular proteins (50 µg) were loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis and immunoblotted with the following antibodies: p53 (sc-126), p21 (sc-397), and PARP (sc-7150) from Santa Cruz Biotechnology (Heidelberg, Germany), γ-H2AX (2577) from Cell Signaling, Danvers, MA, POLA1 (31777) from Abcam (Cambridge, UK), and GAPDH (MAB5476) (Abnova, Heidelberg, Germany).

    Techniques: Staining, TUNEL Assay

    Expression analysis of pUL69 and its betaherpesviral homologs. (A and B) HEK293T cells were transfected with plasmids encoding N-terminally FLAG-tagged (A) or Myc-tagged (B) versions of pUL69 (lane 2), pC69 (lane 3), pRh69 (lane 4), pM69 (lane 5), and pU42 of HHV6 (lane 6) and ElHV1 (lane 7), as indicated. Transfection of an empty vector served as a specificity control (mock, lanes 1). Cells were lysed under denaturing conditions at about 36 h posttransfection. Lysates were separated by SDS-PAGE, and protein expression was analyzed by Western blotting using anti-FLAG- or anti-Myc-specific antibodies. Molecular size markers are indicated (kDa).

    Journal: Journal of Virology

    Article Title: Characterization of the Betaherpesviral pUL69 Protein Family Reveals Binding of the Cellular mRNA Export Factor UAP56 as a Prerequisite for Stimulation of Nuclear mRNA Export and for Efficient Viral Replication ▿

    doi: 10.1128/JVI.01347-10

    Figure Lengend Snippet: Expression analysis of pUL69 and its betaherpesviral homologs. (A and B) HEK293T cells were transfected with plasmids encoding N-terminally FLAG-tagged (A) or Myc-tagged (B) versions of pUL69 (lane 2), pC69 (lane 3), pRh69 (lane 4), pM69 (lane 5), and pU42 of HHV6 (lane 6) and ElHV1 (lane 7), as indicated. Transfection of an empty vector served as a specificity control (mock, lanes 1). Cells were lysed under denaturing conditions at about 36 h posttransfection. Lysates were separated by SDS-PAGE, and protein expression was analyzed by Western blotting using anti-FLAG- or anti-Myc-specific antibodies. Molecular size markers are indicated (kDa).

    Article Snippet: Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 12.5% polyacrylamide gels and transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany), followed by chemiluminescence detection according to the manufacturer's protocol (ECL Western blotting detection kit; Amersham Pharmacia Europe, Freiburg, Germany).

    Techniques: Expressing, Transfection, Plasmid Preparation, SDS Page, Western Blot

    Cellular mRNA 3′-end processing enhances but is not required for PABPC-induced hyperadenylation. (A) HEK 293T cells were transfected with the indicated plasmids for 24 h, and then total RNA was isolated and Northern blotted with 32 P-labeled GFP and 18S probes. (B) Same protocol as described in the legend to panel A, but cells were treated with 5 ng/ml LMB for 12 h prior to RNA isolation to enhance detection of hyperadenylated species. (C) HEK 293T cells were transfected with the indicated plasmids for 24 h and then treated with 5 ng/ml LMB for 12 h. Total RNA was isolated and incubated in the presence or absence of oligo(dT) and then digested with RNaseH. RNA was visualized by Northern blotting with 32 P-labeled GFP and 18S probes. (D) HEK 293T cells were transfected twice over 48 h with either PAPII siRNAs or control siRNAs and then subsequently transfected in duplicate with DNA plasmids expressing GFP-A 60 -HR alone or together with Flag-PABPC1-NRS for 24 h. Samples were treated with 5 ng/ml LMB for 6 h prior to harvesting either protein (top) or RNA (bottom). Protein lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PAPII, Flag, or actin (as a loading control). RNA from each sample was Northern blotted with 32 P-labeled GFP and 18S probes.

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Import of Cytoplasmic Poly(A) Binding Protein Restricts Gene Expression via Hyperadenylation and Nuclear Retention of mRNA ▿

    doi: 10.1128/MCB.00600-10

    Figure Lengend Snippet: Cellular mRNA 3′-end processing enhances but is not required for PABPC-induced hyperadenylation. (A) HEK 293T cells were transfected with the indicated plasmids for 24 h, and then total RNA was isolated and Northern blotted with 32 P-labeled GFP and 18S probes. (B) Same protocol as described in the legend to panel A, but cells were treated with 5 ng/ml LMB for 12 h prior to RNA isolation to enhance detection of hyperadenylated species. (C) HEK 293T cells were transfected with the indicated plasmids for 24 h and then treated with 5 ng/ml LMB for 12 h. Total RNA was isolated and incubated in the presence or absence of oligo(dT) and then digested with RNaseH. RNA was visualized by Northern blotting with 32 P-labeled GFP and 18S probes. (D) HEK 293T cells were transfected twice over 48 h with either PAPII siRNAs or control siRNAs and then subsequently transfected in duplicate with DNA plasmids expressing GFP-A 60 -HR alone or together with Flag-PABPC1-NRS for 24 h. Samples were treated with 5 ng/ml LMB for 6 h prior to harvesting either protein (top) or RNA (bottom). Protein lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PAPII, Flag, or actin (as a loading control). RNA from each sample was Northern blotted with 32 P-labeled GFP and 18S probes.

    Article Snippet: Equivalent micrograms of each sample were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride membrane, and subjected to immunoblotting with either mouse monoclonal GFP (1:2,000 dilution), rabbit polyclonal PABPC 4992 (1:2,000 dilution) (Cell Signaling Technology), or rabbit polyclonal SOX J5803 (1:5,000 dilution) ( ) primary antibodies, followed by incubation with horseradish peroxidase-conjugated actin antibodies (for loading control) and goat anti-mouse or goat anti-rabbit secondary antibodies (1:5,000 dilution) (Southern Biotechnology Associates).

    Techniques: Transfection, Isolation, Northern Blot, Labeling, Incubation, Expressing, SDS Page

    PABPC RRM1 and RRM2 are necessary and sufficient to induce hyperadenylation. (A) Diagram of PABPC, showing the 4 RRMs, followed by the linker region and conserved helical carboxyl terminus (square). (B) Expression levels from the indicated PABPC1 WT and mutant constructs were monitored following transfection into 293T cells for 24 h. Protein lysates harvested, resolved by SDS-PAGE, and immunoblotted with a mixture of anti-Flag and anti-HA antibodies or with antiactin antibody as a loading control. (C and D) HEK 293T cells were transfected with the indicated plasmids for 24 h and then subjected to oligo(dT) in situ hybridization and immunofluorescence assays with anti-HA antibodies. Nuclei were stained with DAPI. (E) HEK 293T cells were transfected with plasmids expressing GFP and either empty vector or the indicated PABPC construct for 24 h. Total RNA was then resolved by agarose-formaldehyde gel electrophoresis and Northern blotted with 32 P-labeled GFP and 18S probes. Hyperadenylated species are indicated by the labeled bracket [hyp(A) GFP].

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Import of Cytoplasmic Poly(A) Binding Protein Restricts Gene Expression via Hyperadenylation and Nuclear Retention of mRNA ▿

    doi: 10.1128/MCB.00600-10

    Figure Lengend Snippet: PABPC RRM1 and RRM2 are necessary and sufficient to induce hyperadenylation. (A) Diagram of PABPC, showing the 4 RRMs, followed by the linker region and conserved helical carboxyl terminus (square). (B) Expression levels from the indicated PABPC1 WT and mutant constructs were monitored following transfection into 293T cells for 24 h. Protein lysates harvested, resolved by SDS-PAGE, and immunoblotted with a mixture of anti-Flag and anti-HA antibodies or with antiactin antibody as a loading control. (C and D) HEK 293T cells were transfected with the indicated plasmids for 24 h and then subjected to oligo(dT) in situ hybridization and immunofluorescence assays with anti-HA antibodies. Nuclei were stained with DAPI. (E) HEK 293T cells were transfected with plasmids expressing GFP and either empty vector or the indicated PABPC construct for 24 h. Total RNA was then resolved by agarose-formaldehyde gel electrophoresis and Northern blotted with 32 P-labeled GFP and 18S probes. Hyperadenylated species are indicated by the labeled bracket [hyp(A) GFP].

    Article Snippet: Equivalent micrograms of each sample were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride membrane, and subjected to immunoblotting with either mouse monoclonal GFP (1:2,000 dilution), rabbit polyclonal PABPC 4992 (1:2,000 dilution) (Cell Signaling Technology), or rabbit polyclonal SOX J5803 (1:5,000 dilution) ( ) primary antibodies, followed by incubation with horseradish peroxidase-conjugated actin antibodies (for loading control) and goat anti-mouse or goat anti-rabbit secondary antibodies (1:5,000 dilution) (Southern Biotechnology Associates).

    Techniques: Expressing, Mutagenesis, Construct, Transfection, SDS Page, In Situ Hybridization, Immunofluorescence, Staining, Plasmid Preparation, Nucleic Acid Electrophoresis, Northern Blot, Labeling

    PABPC4 is induced upon PABPC1 depletion and is directed to the nucleus by SOX. (A) COS7, HEK 293T, and HeLa cells were transfected with either control siRNAs (si) or 2 independent siRNAs specific for PABPC1 (PABPC1#1 and PABPC1#2). siRNA transfections for COS7 and HEK 293T cells were performed in duplicate. At 72 h posttransfection, cell lysates were resolved by SDS-PAGE and immunoblotted with anti-PABPC antibodies. (B) HeLa and HEK 293T cells were transfected with control siRNAs or the indicated siRNAs specific for PABPC1 and/or PABPC4. Two independent PABPC4-specific siRNAs were used (PABPC4#1 and PABPC4#2). Lysates were then harvested and immunoblotted as described in the legend to panel A. (C) HEK 293T cells were transfected with a plasmid expressing HA-tagged PABPC4 alone or together with a plasmid expressing SOX and, 24 h later, subjected to immunofluorescence assays with anti-HA and anti-SOX antibodies. DAPI staining was used to visualize nuclei.

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Import of Cytoplasmic Poly(A) Binding Protein Restricts Gene Expression via Hyperadenylation and Nuclear Retention of mRNA ▿

    doi: 10.1128/MCB.00600-10

    Figure Lengend Snippet: PABPC4 is induced upon PABPC1 depletion and is directed to the nucleus by SOX. (A) COS7, HEK 293T, and HeLa cells were transfected with either control siRNAs (si) or 2 independent siRNAs specific for PABPC1 (PABPC1#1 and PABPC1#2). siRNA transfections for COS7 and HEK 293T cells were performed in duplicate. At 72 h posttransfection, cell lysates were resolved by SDS-PAGE and immunoblotted with anti-PABPC antibodies. (B) HeLa and HEK 293T cells were transfected with control siRNAs or the indicated siRNAs specific for PABPC1 and/or PABPC4. Two independent PABPC4-specific siRNAs were used (PABPC4#1 and PABPC4#2). Lysates were then harvested and immunoblotted as described in the legend to panel A. (C) HEK 293T cells were transfected with a plasmid expressing HA-tagged PABPC4 alone or together with a plasmid expressing SOX and, 24 h later, subjected to immunofluorescence assays with anti-HA and anti-SOX antibodies. DAPI staining was used to visualize nuclei.

    Article Snippet: Equivalent micrograms of each sample were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride membrane, and subjected to immunoblotting with either mouse monoclonal GFP (1:2,000 dilution), rabbit polyclonal PABPC 4992 (1:2,000 dilution) (Cell Signaling Technology), or rabbit polyclonal SOX J5803 (1:5,000 dilution) ( ) primary antibodies, followed by incubation with horseradish peroxidase-conjugated actin antibodies (for loading control) and goat anti-mouse or goat anti-rabbit secondary antibodies (1:5,000 dilution) (Southern Biotechnology Associates).

    Techniques: Transfection, SDS Page, Plasmid Preparation, Expressing, Immunofluorescence, Staining

    Nuclear accumulation of PABPC causes mRNA retention and hyperadenylation. (A) HEK 293T cells were transfected with the indicated PABPC1 expression plasmids for 24 h. Lysates were then harvested, resolved by SDS-PAGE, and immunoblotted with anti-PABPC antibodies to detect both endogenous (bottom arrow) and exogenous (upper arrow) PABPC proteins. Note that Flag-PABPC1 comigrates with endogenous PABPC1 in this blot. In parallel, antiactin immunoblotting was performed to control for loading. (B) HEK 293T cells were transfected as described in the legend to panel A for 24 h and then processed for oligo(dT) in situ hybridization, followed by immunofluorescence assays with anti-Flag antibodies. (C) HEK 293T cells were cotransfected with the indicated plasmids for 24 h. After treatment with leptomycin B (LMB) for 12 h to stabilize hyperadenylated species, total RNA and proteins were isolated. Total RNA was incubated in the presence or absence of oligo(dT) and digested with RNaseH. Products were resolved on a 1.2% agarose-formaldehyde gel and Northern blotted with 32 P-labeled GFP and 18S probes (top). Hyperadenylated species are indicated by the labeled bracket [hyp(A) GFP]. Total protein was resolved by SDS-PAGE and immunoblotted using anti-Flag and anti-SOX antibodies (bottom). Actin served as a loading control. (D) Cells were transfected with the indicated plasmid as described above and treated with LMB for 7 h. Total RNA was then isolated from whole cells (W) or cytoplasmic fractions (C) and nuclear fractions (N) fractions and Northern blotted with 32 P-labeled GFP and 18S probes. Hyperadenylated species are indicated by the labeled bracket [hyp(A) GFP].

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Import of Cytoplasmic Poly(A) Binding Protein Restricts Gene Expression via Hyperadenylation and Nuclear Retention of mRNA ▿

    doi: 10.1128/MCB.00600-10

    Figure Lengend Snippet: Nuclear accumulation of PABPC causes mRNA retention and hyperadenylation. (A) HEK 293T cells were transfected with the indicated PABPC1 expression plasmids for 24 h. Lysates were then harvested, resolved by SDS-PAGE, and immunoblotted with anti-PABPC antibodies to detect both endogenous (bottom arrow) and exogenous (upper arrow) PABPC proteins. Note that Flag-PABPC1 comigrates with endogenous PABPC1 in this blot. In parallel, antiactin immunoblotting was performed to control for loading. (B) HEK 293T cells were transfected as described in the legend to panel A for 24 h and then processed for oligo(dT) in situ hybridization, followed by immunofluorescence assays with anti-Flag antibodies. (C) HEK 293T cells were cotransfected with the indicated plasmids for 24 h. After treatment with leptomycin B (LMB) for 12 h to stabilize hyperadenylated species, total RNA and proteins were isolated. Total RNA was incubated in the presence or absence of oligo(dT) and digested with RNaseH. Products were resolved on a 1.2% agarose-formaldehyde gel and Northern blotted with 32 P-labeled GFP and 18S probes (top). Hyperadenylated species are indicated by the labeled bracket [hyp(A) GFP]. Total protein was resolved by SDS-PAGE and immunoblotted using anti-Flag and anti-SOX antibodies (bottom). Actin served as a loading control. (D) Cells were transfected with the indicated plasmid as described above and treated with LMB for 7 h. Total RNA was then isolated from whole cells (W) or cytoplasmic fractions (C) and nuclear fractions (N) fractions and Northern blotted with 32 P-labeled GFP and 18S probes. Hyperadenylated species are indicated by the labeled bracket [hyp(A) GFP].

    Article Snippet: Equivalent micrograms of each sample were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride membrane, and subjected to immunoblotting with either mouse monoclonal GFP (1:2,000 dilution), rabbit polyclonal PABPC 4992 (1:2,000 dilution) (Cell Signaling Technology), or rabbit polyclonal SOX J5803 (1:5,000 dilution) ( ) primary antibodies, followed by incubation with horseradish peroxidase-conjugated actin antibodies (for loading control) and goat anti-mouse or goat anti-rabbit secondary antibodies (1:5,000 dilution) (Southern Biotechnology Associates).

    Techniques: Transfection, Expressing, SDS Page, In Situ Hybridization, Immunofluorescence, Isolation, Incubation, Northern Blot, Labeling, Plasmid Preparation

    Cytoplasmic PABPC and nuclear PABPC have opposing effects on gene expression. (A, B) HEK 293T cells were transfected with GFP and increasing amounts of Flag-PABPC1-CRS or Flag-PABPC1-NRS (100 to 900 ng) for 24 h. Total RNA was then isolated and visualized by Northern blotting with 32 P-labeled GFP and 18S probes. Hyperadenylated species are indicated by the labeled bracket [hyp(A) GFP]. To monitor protein levels, protein lysate from the transfected cells was resolved by SDS-PAGE and immunoblotted with anti-Flag or antiactin (loading control) antibodies. (C) HEK 293T cells were transfected with GFP and empty vector, Flag-PABPC1-CRS, or Flag-PABPC1-NRS. At 24 h posttransfection, 5 μg/ml of actinomycin D was added to block transcription, and total RNA was harvested at the indicated time points thereafter. RNA was then visualized by Northern blotting with GFP and 18S probes, and the GFP mRNA half-life was calculated after 18S normalization. Error bars indicate standard errors between samples. Data were derived from five independent experiments. (D) HEK 293T cells were transfected with plasmids expressing GFP and empty vector, Flag-PABPC1-NRS, or Flag-PABPC1-CRS for 24 h. Equivalent amounts of protein lysate were resolved by SDS-PAGE and immunoblotted with antibodies against GFP, Flag, and actin (as a loading control).

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Import of Cytoplasmic Poly(A) Binding Protein Restricts Gene Expression via Hyperadenylation and Nuclear Retention of mRNA ▿

    doi: 10.1128/MCB.00600-10

    Figure Lengend Snippet: Cytoplasmic PABPC and nuclear PABPC have opposing effects on gene expression. (A, B) HEK 293T cells were transfected with GFP and increasing amounts of Flag-PABPC1-CRS or Flag-PABPC1-NRS (100 to 900 ng) for 24 h. Total RNA was then isolated and visualized by Northern blotting with 32 P-labeled GFP and 18S probes. Hyperadenylated species are indicated by the labeled bracket [hyp(A) GFP]. To monitor protein levels, protein lysate from the transfected cells was resolved by SDS-PAGE and immunoblotted with anti-Flag or antiactin (loading control) antibodies. (C) HEK 293T cells were transfected with GFP and empty vector, Flag-PABPC1-CRS, or Flag-PABPC1-NRS. At 24 h posttransfection, 5 μg/ml of actinomycin D was added to block transcription, and total RNA was harvested at the indicated time points thereafter. RNA was then visualized by Northern blotting with GFP and 18S probes, and the GFP mRNA half-life was calculated after 18S normalization. Error bars indicate standard errors between samples. Data were derived from five independent experiments. (D) HEK 293T cells were transfected with plasmids expressing GFP and empty vector, Flag-PABPC1-NRS, or Flag-PABPC1-CRS for 24 h. Equivalent amounts of protein lysate were resolved by SDS-PAGE and immunoblotted with antibodies against GFP, Flag, and actin (as a loading control).

    Article Snippet: Equivalent micrograms of each sample were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride membrane, and subjected to immunoblotting with either mouse monoclonal GFP (1:2,000 dilution), rabbit polyclonal PABPC 4992 (1:2,000 dilution) (Cell Signaling Technology), or rabbit polyclonal SOX J5803 (1:5,000 dilution) ( ) primary antibodies, followed by incubation with horseradish peroxidase-conjugated actin antibodies (for loading control) and goat anti-mouse or goat anti-rabbit secondary antibodies (1:5,000 dilution) (Southern Biotechnology Associates).

    Techniques: Expressing, Transfection, Isolation, Northern Blot, Labeling, SDS Page, Plasmid Preparation, Blocking Assay, Derivative Assay