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  • 99
    Thermo Fisher polyvinylidene difluoride pvdf membranes
    Deletion of the C-terminal domain increases auto-phosphorylation activity. ( A ) Partial tryptic digestion of recombinant MPK10. 50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at the indicated time points and the reaction was stopped either by adding Laemmli buffer (for N-terminal sequencing) or by lowering the pH to 5.0 and subsequent freezing (for mass determination by SELDI-TOF). For N-terminal sequencing, samples were separated by <t>SDS-PAGE,</t> transferred on <t>PVDF</t> membrane and stained by amidoblack. N-terminal sequencing was performed at the protein analysis platform at the Institut Pasteur. For mass determination, samples were immobilized on a H4 ProteinChip Array (C16 reversed phase surface) and peptide masses identified by SELDI-TOF. Results of the N-terminal sequencing are represented by the cartoon in ( B ), and the sequences are indicated in ( C ). Italic characters represent the Strep3-tag and bold characters represent the sequence of Leishmania major MPK10. White and grey arrowheads indicate respectively lysine or arginine residues recognized by trypsine, including K12, K24, K30 and R392. The white arrow at the position D387 indicates the position of the last cleaved residue resulting in the generation of the form lacking the last 46 amino acids of MPK10. ( D ) In vitro kinase assay using recombinant His-MPK10 (NM) and the truncated kinase mutants His-MPK10-ΔC (ΔC), and His-MPK10-ΔC_K51A (ΔC_K/A). Results are representative of three independent experiments. Purified proteins were incubated with four different substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed at the same time for 30 min at pH 7.5 and 37°C and reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (right), and signals were revealed by auto-radiography with the same exposure time between the different gels (left). The brackets in (D) indicate auto-phosphorylation (Auto-P) and substrate phosphorylation (Substrate-P) signals.
    Polyvinylidene Difluoride Pvdf Membranes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore polyvinylidene fluoride pvdf membrane
    Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4. The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by <t>SDS-PAGE</t> under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto <t>PVDF</t> membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.
    Polyvinylidene Fluoride Pvdf Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher pvdf immobilon p
    Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4. The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by <t>SDS-PAGE</t> under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto <t>PVDF</t> membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.
    Pvdf Immobilon P, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bio-Rad polyvinyldifluoride pvdf filter
    Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4. The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by <t>SDS-PAGE</t> under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto <t>PVDF</t> membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.
    Polyvinyldifluoride Pvdf Filter, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore immobilon p polyvinylidene difluoride
    Effect of CSE on HSA Cys34 free sulfhydryl group as determined by the Ellman assay. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% (v/v) CSE and then exhaustively dialyzed. The concentration of Cys34 sulfhydryl groups in HSA samples was determined by the Ellman assay at 412 nm as described under Materials and methods . Data are presented as the mean ± SD of three independent measurements. Inset: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by biotin-HPDP binding and Western blot analysis. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% CSE, exhaustively dialyzed and then labeled at Cys34 with biotin-HPDP as described under Materials and methods . Proteins (10 µg/lane) were separated by SDS-PAGE and biotin-HPDP binding was detected by Western blot analysis using streptavidin-HRP as described under Materials and methods (immunoblot inset). Amido Black staining of the same <t>PVDF</t> membrane showed equal protein loading and transfer (not shown). Immunoblot shown is representative of three independent determinations. Bar-graph inset shows densitometric analysis of biotin-HPDP incorporation. Data are presented as the mean ± SD of three independent determinations.
    Immobilon P Polyvinylidene Difluoride, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 909 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare immobilon p pvdf membrane
    Western blotting confirms the presence of ApoE and Na + /K + -ATPase α-chains in SAF preparations. Based on an estimate of 10 µg PrP Sc per brain, the equivalent of 0.25 µg/µl PrP Sc from an ME7 (lane 1), 22F (lane 2) and 79A (lane 3) SAF preparation were resolved by SDS-PAGE, blotted onto <t>PVDF</t> membrane and probed with the primary antibody against (A) Apolipoprotein E (B) Total Na + /K + ATPase α-chains using a pan α-chain antibody (C) Na + /K + ATPase α2 isoform and (D) Na + /K + ATPase α3 isoform. In all cases, in lanes 4 and 5 were loaded the equivalent of 0.25 µg/µl of control preparation material from uninfected WT and PrP −/− mouse brains respectively. Molecular weight markers are in kDa.
    Immobilon P Pvdf Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche polyvinylidene difluoride pvdf membranes
    In vitro cleavage of WT vSag7 or the CS12X triple mutant by cathepsin L. Equal amounts of purified [ 35 S]methionine-labeled proteins produced with a coupled in vitro transcription-translation system were subjected to cleavage with 10 ng of purified cathepsin L, fractionated by <t>SDS–15%</t> PAGE, transferred to <t>PVDF</t> membranes, and exposed to a PhosphorImager screen. Time of cleavage is in minutes. These data are representative of three independent experiments. The values on the left are the molecular masses in kilodaltons.
    Polyvinylidene Difluoride Pvdf Membranes, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 629 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PerkinElmer polyvinylidene difluoride pvdf membrane
    In vitro cleavage of WT vSag7 or the CS12X triple mutant by cathepsin L. Equal amounts of purified [ 35 S]methionine-labeled proteins produced with a coupled in vitro transcription-translation system were subjected to cleavage with 10 ng of purified cathepsin L, fractionated by <t>SDS–15%</t> PAGE, transferred to <t>PVDF</t> membranes, and exposed to a PhosphorImager screen. Time of cleavage is in minutes. These data are representative of three independent experiments. The values on the left are the molecular masses in kilodaltons.
    Polyvinylidene Difluoride Pvdf Membrane, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad polyvinylidene fluoride pvdf membrane
    The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to <t>PVDF</t> membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. <t>Immunoblot</t> of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).
    Polyvinylidene Fluoride Pvdf Membrane, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 3964 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA polyvinylidene fluoride pvdf membrane
    Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by <t>SDS–PAGE</t> and transferred onto <t>PVDF</t> membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.
    Polyvinylidene Fluoride Pvdf Membrane, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 766 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck & Co pvdf membrane immobilon p
    Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by <t>SDS–PAGE</t> and transferred onto <t>PVDF</t> membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.
    Pvdf Membrane Immobilon P, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore immobilon p hydrophobic pvdf plates
    Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by <t>SDS–PAGE</t> and transferred onto <t>PVDF</t> membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.
    Immobilon P Hydrophobic Pvdf Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore enzymatic
    Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by <t>SDS–PAGE</t> and transferred onto <t>PVDF</t> membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.
    Enzymatic, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad polyvinylidene difluoride pvdf
    <t>Immunoblot</t> analysis of r-OspF protein family members expressed in E. coli . Each member of the ospF gene family was cloned and expressed as an S-tag fusion protein using ligase-independent cloning methods as described in the text. Proteins from E. coli cultures that were induced to express the r-proteins with IPTG were fractionated by SDS-PAGE and transferred to a <t>PVDF</t> membrane by electroblotting. The membrane on the left was screened with anti-S-Tag protein HRP conjugate, while the membrane on the right was screened with a polyclonal anti-OspF antiserum (generated with a gene of B. burgdorferi N40 origin). Immunoblot methods are described in the text. Molecular size standards are indicated on the left.
    Polyvinylidene Difluoride Pvdf, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Fisher Scientific immobilon polyvinyldifluoride pvdf membranes
    <t>Immunoblot</t> analysis of r-OspF protein family members expressed in E. coli . Each member of the ospF gene family was cloned and expressed as an S-tag fusion protein using ligase-independent cloning methods as described in the text. Proteins from E. coli cultures that were induced to express the r-proteins with IPTG were fractionated by SDS-PAGE and transferred to a <t>PVDF</t> membrane by electroblotting. The membrane on the left was screened with anti-S-Tag protein HRP conjugate, while the membrane on the right was screened with a polyclonal anti-OspF antiserum (generated with a gene of B. burgdorferi N40 origin). Immunoblot methods are described in the text. Molecular size standards are indicated on the left.
    Immobilon Polyvinyldifluoride Pvdf Membranes, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare polyvinylidene fluoride pvdf
    <t>Immunoblot</t> analysis of r-OspF protein family members expressed in E. coli . Each member of the ospF gene family was cloned and expressed as an S-tag fusion protein using ligase-independent cloning methods as described in the text. Proteins from E. coli cultures that were induced to express the r-proteins with IPTG were fractionated by SDS-PAGE and transferred to a <t>PVDF</t> membrane by electroblotting. The membrane on the left was screened with anti-S-Tag protein HRP conjugate, while the membrane on the right was screened with a polyclonal anti-OspF antiserum (generated with a gene of B. burgdorferi N40 origin). Immunoblot methods are described in the text. Molecular size standards are indicated on the left.
    Polyvinylidene Fluoride Pvdf, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Deletion of the C-terminal domain increases auto-phosphorylation activity. ( A ) Partial tryptic digestion of recombinant MPK10. 50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at the indicated time points and the reaction was stopped either by adding Laemmli buffer (for N-terminal sequencing) or by lowering the pH to 5.0 and subsequent freezing (for mass determination by SELDI-TOF). For N-terminal sequencing, samples were separated by SDS-PAGE, transferred on PVDF membrane and stained by amidoblack. N-terminal sequencing was performed at the protein analysis platform at the Institut Pasteur. For mass determination, samples were immobilized on a H4 ProteinChip Array (C16 reversed phase surface) and peptide masses identified by SELDI-TOF. Results of the N-terminal sequencing are represented by the cartoon in ( B ), and the sequences are indicated in ( C ). Italic characters represent the Strep3-tag and bold characters represent the sequence of Leishmania major MPK10. White and grey arrowheads indicate respectively lysine or arginine residues recognized by trypsine, including K12, K24, K30 and R392. The white arrow at the position D387 indicates the position of the last cleaved residue resulting in the generation of the form lacking the last 46 amino acids of MPK10. ( D ) In vitro kinase assay using recombinant His-MPK10 (NM) and the truncated kinase mutants His-MPK10-ΔC (ΔC), and His-MPK10-ΔC_K51A (ΔC_K/A). Results are representative of three independent experiments. Purified proteins were incubated with four different substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed at the same time for 30 min at pH 7.5 and 37°C and reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (right), and signals were revealed by auto-radiography with the same exposure time between the different gels (left). The brackets in (D) indicate auto-phosphorylation (Auto-P) and substrate phosphorylation (Substrate-P) signals.

    Journal: PLoS Pathogens

    Article Title: Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

    doi: 10.1371/journal.ppat.1004347

    Figure Lengend Snippet: Deletion of the C-terminal domain increases auto-phosphorylation activity. ( A ) Partial tryptic digestion of recombinant MPK10. 50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at the indicated time points and the reaction was stopped either by adding Laemmli buffer (for N-terminal sequencing) or by lowering the pH to 5.0 and subsequent freezing (for mass determination by SELDI-TOF). For N-terminal sequencing, samples were separated by SDS-PAGE, transferred on PVDF membrane and stained by amidoblack. N-terminal sequencing was performed at the protein analysis platform at the Institut Pasteur. For mass determination, samples were immobilized on a H4 ProteinChip Array (C16 reversed phase surface) and peptide masses identified by SELDI-TOF. Results of the N-terminal sequencing are represented by the cartoon in ( B ), and the sequences are indicated in ( C ). Italic characters represent the Strep3-tag and bold characters represent the sequence of Leishmania major MPK10. White and grey arrowheads indicate respectively lysine or arginine residues recognized by trypsine, including K12, K24, K30 and R392. The white arrow at the position D387 indicates the position of the last cleaved residue resulting in the generation of the form lacking the last 46 amino acids of MPK10. ( D ) In vitro kinase assay using recombinant His-MPK10 (NM) and the truncated kinase mutants His-MPK10-ΔC (ΔC), and His-MPK10-ΔC_K51A (ΔC_K/A). Results are representative of three independent experiments. Purified proteins were incubated with four different substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed at the same time for 30 min at pH 7.5 and 37°C and reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (right), and signals were revealed by auto-radiography with the same exposure time between the different gels (left). The brackets in (D) indicate auto-phosphorylation (Auto-P) and substrate phosphorylation (Substrate-P) signals.

    Article Snippet: Alternatively, proteins were separated by SDS–PAGE on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and blotted onto polyvinylidene difluoride (PVDF) membranes (Pierce).

    Techniques: Activity Assay, Recombinant, Sequencing, SDS Page, Staining, In Vitro, Kinase Assay, Purification, Incubation, Positive Control

    Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4. The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by SDS-PAGE under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto PVDF membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.

    Journal: PLoS ONE

    Article Title: Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4

    doi: 10.1371/journal.pone.0133784

    Figure Lengend Snippet: Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4. The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by SDS-PAGE under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto PVDF membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.

    Article Snippet: The protein components resolved by SDS-PAGE were electrophoretically blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA), and then the reactivity of the transferred proteins with primary Abs (0.5–1 μg/ml) or several types of biotin-labeled lectins was assayed using HRP-conjugated secondary antibodies or streptavidin and an ECL Western blotting detection system (GE Healthcare, Buckinghamshire, UK) [ , ].

    Techniques: Immunoprecipitation, SDS Page, Positive Control, Whole Genome Amplification

    Immunoreactivity of Ts4 against testicular proteins pretreated with periodic acid. The testicular TS fractions (each 5 μg protein) were loaded on a 10% gel, separated by SDS-PAGE under reducing conditions, and then blotted onto a PVDF membrane. The PVDF membrane was divided into individual lanes, which were treated with 0.075 M NaIO 4 and HIO 4 2H 2 O in PBS for various times. Specific bands were detected with Ts4 or 6035 (arrowheads). Mr, molecular mass.

    Journal: PLoS ONE

    Article Title: Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4

    doi: 10.1371/journal.pone.0133784

    Figure Lengend Snippet: Immunoreactivity of Ts4 against testicular proteins pretreated with periodic acid. The testicular TS fractions (each 5 μg protein) were loaded on a 10% gel, separated by SDS-PAGE under reducing conditions, and then blotted onto a PVDF membrane. The PVDF membrane was divided into individual lanes, which were treated with 0.075 M NaIO 4 and HIO 4 2H 2 O in PBS for various times. Specific bands were detected with Ts4 or 6035 (arrowheads). Mr, molecular mass.

    Article Snippet: The protein components resolved by SDS-PAGE were electrophoretically blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA), and then the reactivity of the transferred proteins with primary Abs (0.5–1 μg/ml) or several types of biotin-labeled lectins was assayed using HRP-conjugated secondary antibodies or streptavidin and an ECL Western blotting detection system (GE Healthcare, Buckinghamshire, UK) [ , ].

    Techniques: SDS Page

    SDS-PAGE analyses of mouse testicular proteins immunoprecipitated with Ts4. Western blot analyses using Ts4 (A). Testicular TS proteins immunoprecipitated with either Ts4 or normal control mouse IgM (n.c.) were separated via SDS-PAGE on 10% gels under reducing conditions. Control experiments were conducted under the same conditions, but in the absence of the testicular extract (buf). Separated proteins were electroblotted onto PVDF membranes and then probed with Ts4. Arrowheads indicate molecular mass (Mr) of the specific immunoreactive bands. Visualized by CBB-staining (B). The same samples were applied to lanes of the 10% SDS-PAGE gel under reducing conditions, and then the gel was CBB-stained. Apparent positions of dominant bands obtained via immunoprecipitation with Ts4 are indicated with arrowheads.

    Journal: PLoS ONE

    Article Title: Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4

    doi: 10.1371/journal.pone.0133784

    Figure Lengend Snippet: SDS-PAGE analyses of mouse testicular proteins immunoprecipitated with Ts4. Western blot analyses using Ts4 (A). Testicular TS proteins immunoprecipitated with either Ts4 or normal control mouse IgM (n.c.) were separated via SDS-PAGE on 10% gels under reducing conditions. Control experiments were conducted under the same conditions, but in the absence of the testicular extract (buf). Separated proteins were electroblotted onto PVDF membranes and then probed with Ts4. Arrowheads indicate molecular mass (Mr) of the specific immunoreactive bands. Visualized by CBB-staining (B). The same samples were applied to lanes of the 10% SDS-PAGE gel under reducing conditions, and then the gel was CBB-stained. Apparent positions of dominant bands obtained via immunoprecipitation with Ts4 are indicated with arrowheads.

    Article Snippet: The protein components resolved by SDS-PAGE were electrophoretically blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA), and then the reactivity of the transferred proteins with primary Abs (0.5–1 μg/ml) or several types of biotin-labeled lectins was assayed using HRP-conjugated secondary antibodies or streptavidin and an ECL Western blotting detection system (GE Healthcare, Buckinghamshire, UK) [ , ].

    Techniques: SDS Page, Immunoprecipitation, Western Blot, Staining

    Bay11-7085 induced apoptosis is caspase dependent in PEL cell lines. ( A ) Activation of caspases-9, -3, and cleavage of PARP induced by Bay11-7085 treatment in PEL cells. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-9, caspase-3, cleaved caspase-3 and PARP. Beta-actin was used for equal loading. ( B ) Bay11-7085 treatment causes cleavage of caspase-8 and truncation of Bid in PEL cells. After treatment with 5 and 10 µM Bay11-7085 for 24 hours, cells were lysed, and equal amount of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-8 and Bid. ( C, D and E ) Bay11-7085-induced apoptosis is caspase dependent in PEL cells. PEL cells were pre-treated with 80 µM zVAD-fmk for 2 hours and then treated with 10 µM Bay11-7085 for 24 hours. Following treatment, cells were either lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-3, cleaved caspase-3 and PARP ( C ) or stained with FITC conjugated annexin V/PI and analyzed by flow cytometry ( D ). Bar graph denotes percentage apoptosis from three independent experiments ( E ).

    Journal: PLoS ONE

    Article Title: Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    doi: 10.1371/journal.pone.0039945

    Figure Lengend Snippet: Bay11-7085 induced apoptosis is caspase dependent in PEL cell lines. ( A ) Activation of caspases-9, -3, and cleavage of PARP induced by Bay11-7085 treatment in PEL cells. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-9, caspase-3, cleaved caspase-3 and PARP. Beta-actin was used for equal loading. ( B ) Bay11-7085 treatment causes cleavage of caspase-8 and truncation of Bid in PEL cells. After treatment with 5 and 10 µM Bay11-7085 for 24 hours, cells were lysed, and equal amount of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-8 and Bid. ( C, D and E ) Bay11-7085-induced apoptosis is caspase dependent in PEL cells. PEL cells were pre-treated with 80 µM zVAD-fmk for 2 hours and then treated with 10 µM Bay11-7085 for 24 hours. Following treatment, cells were either lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-3, cleaved caspase-3 and PARP ( C ) or stained with FITC conjugated annexin V/PI and analyzed by flow cytometry ( D ). Bar graph denotes percentage apoptosis from three independent experiments ( E ).

    Article Snippet: 10–15 µg of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) (Immobilion, Millipore).

    Techniques: Activation Assay, SDS Page, Staining, Flow Cytometry, Cytometry

    Bay11-7085 treatment of PEL cells activates mitochondrial apoptotic pathway in PEL cell lines. ( A ) Bay11-7085-induced Bax activation in PEL cells. After treating with 10 µM Bay11-7085 for indicated time periods, BC1 cells were lysed in 1% Chaps lysis buffer and subjected to immuno-precipitation with anti-Bax 6A7 antibody for detection of conformationally changed Bax protein. In addition, the total cell lysates were applied directly to SDS–PAGE, transferred to immobilon membrane and immuno-blotted with specific anti-Bax polyclonal antibody. ( B ) Bay11-7085 treatment causes change in mitochondrial membrane potential in PEL cells. PEL cells were treated with and without 5 and 10 µM Bay11-7085 for 24 hours. Live cells with intact mitochondrial membrane potential and dead cells with lost mitochondrial membrane potential was measured by JC-1 staining and analyzed by flow cytometry as described in Materials and Methods . ( C ) Bay11-7085 treatment causes release of cytochrome c from mitochondria into cytosole in PEL cells. BC1 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Mitochondrial free cytosolic fractions and cytosolic extracts were isolated and immunoblotted with antibody against cytochrome c and Beta-actin. ( D ) Bay11-7085 treatment causes down-regulation of IAPs in PEL cells. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against cIAP1 and cIAP2. Beat actin was used for equal loading.

    Journal: PLoS ONE

    Article Title: Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    doi: 10.1371/journal.pone.0039945

    Figure Lengend Snippet: Bay11-7085 treatment of PEL cells activates mitochondrial apoptotic pathway in PEL cell lines. ( A ) Bay11-7085-induced Bax activation in PEL cells. After treating with 10 µM Bay11-7085 for indicated time periods, BC1 cells were lysed in 1% Chaps lysis buffer and subjected to immuno-precipitation with anti-Bax 6A7 antibody for detection of conformationally changed Bax protein. In addition, the total cell lysates were applied directly to SDS–PAGE, transferred to immobilon membrane and immuno-blotted with specific anti-Bax polyclonal antibody. ( B ) Bay11-7085 treatment causes change in mitochondrial membrane potential in PEL cells. PEL cells were treated with and without 5 and 10 µM Bay11-7085 for 24 hours. Live cells with intact mitochondrial membrane potential and dead cells with lost mitochondrial membrane potential was measured by JC-1 staining and analyzed by flow cytometry as described in Materials and Methods . ( C ) Bay11-7085 treatment causes release of cytochrome c from mitochondria into cytosole in PEL cells. BC1 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Mitochondrial free cytosolic fractions and cytosolic extracts were isolated and immunoblotted with antibody against cytochrome c and Beta-actin. ( D ) Bay11-7085 treatment causes down-regulation of IAPs in PEL cells. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against cIAP1 and cIAP2. Beat actin was used for equal loading.

    Article Snippet: 10–15 µg of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) (Immobilion, Millipore).

    Techniques: Activation Assay, Lysis, Immunoprecipitation, SDS Page, Staining, Flow Cytometry, Cytometry, Isolation

    Cross-talk between NFκB and PI3-kinase/AKT pathway in PEL cell lines. ( A ) Bay11-7085 treatment inactivates AKT and its down-stream targets in PEL cells. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-FOXO1, p-GSK3, p-Bad and Beta-actin. ( B ) Transcriptional knock down of p65 causes in-activation of AKT and its down-stream targets in PEL cells. BC1 and BC3 cells were transfected with siRNA against p65 for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-FOXO1, p-GSK3, p-Bad and Beta-actin. ( C ) Transcriptional targeting of AKT causes in-activation of NFκB pathway. BC1 cells were transfected with siRNA against AKT for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-IκBα and Beta-actin.

    Journal: PLoS ONE

    Article Title: Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    doi: 10.1371/journal.pone.0039945

    Figure Lengend Snippet: Cross-talk between NFκB and PI3-kinase/AKT pathway in PEL cell lines. ( A ) Bay11-7085 treatment inactivates AKT and its down-stream targets in PEL cells. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-FOXO1, p-GSK3, p-Bad and Beta-actin. ( B ) Transcriptional knock down of p65 causes in-activation of AKT and its down-stream targets in PEL cells. BC1 and BC3 cells were transfected with siRNA against p65 for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-FOXO1, p-GSK3, p-Bad and Beta-actin. ( C ) Transcriptional targeting of AKT causes in-activation of NFκB pathway. BC1 cells were transfected with siRNA against AKT for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-IκBα and Beta-actin.

    Article Snippet: 10–15 µg of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) (Immobilion, Millipore).

    Techniques: SDS Page, Activation Assay, Transfection

    Role of NFκB in PEL cell lines (A) Constitutive expression of NFkB in PEL cells. Nuclear extracts from BC1, BC3, BCBL1 and HBL6 cell lines were prepared as described in material and Methods and electrophoretic mobility shift assay (EMSA) was performed as described in Materials and Methods . Briefly, 5×10 6 cells were washed with cold PBS and suspended in 0.4 mL hypotonic lysis buffer containing protease inhibitors for 30 minutes. The cells were then lysed with 10% Nonidet P-40. ( B ) Bay11-7085 inhibits constitutive nuclear NFkB in PEL cells. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Nuclear extracts were prepared and EMSA was performed. ( C ) Effect of Bay11-7085 on IκBα phosphorylation in PEL cells. BC1 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against phospho-IκBα and Beta actin as indicated. ( D ) Bay11-7085 treatment causes down-regulation of expression of down-stream targets of p65. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against IκBα, Bcl-2, Bcl-Xl, XIAP, Survivin and Beta-actin. ( E ) Transcriptional down-regulation of p65 causes decreased expression of p65 targets in PEL cells. BC1 and BC3 cells were transfected with siRNA against p65 for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against IκBα, Bcl-2, Bcl-Xl, XIAP, Survivin and Beta-actin.

    Journal: PLoS ONE

    Article Title: Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    doi: 10.1371/journal.pone.0039945

    Figure Lengend Snippet: Role of NFκB in PEL cell lines (A) Constitutive expression of NFkB in PEL cells. Nuclear extracts from BC1, BC3, BCBL1 and HBL6 cell lines were prepared as described in material and Methods and electrophoretic mobility shift assay (EMSA) was performed as described in Materials and Methods . Briefly, 5×10 6 cells were washed with cold PBS and suspended in 0.4 mL hypotonic lysis buffer containing protease inhibitors for 30 minutes. The cells were then lysed with 10% Nonidet P-40. ( B ) Bay11-7085 inhibits constitutive nuclear NFkB in PEL cells. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Nuclear extracts were prepared and EMSA was performed. ( C ) Effect of Bay11-7085 on IκBα phosphorylation in PEL cells. BC1 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against phospho-IκBα and Beta actin as indicated. ( D ) Bay11-7085 treatment causes down-regulation of expression of down-stream targets of p65. BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against IκBα, Bcl-2, Bcl-Xl, XIAP, Survivin and Beta-actin. ( E ) Transcriptional down-regulation of p65 causes decreased expression of p65 targets in PEL cells. BC1 and BC3 cells were transfected with siRNA against p65 for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against IκBα, Bcl-2, Bcl-Xl, XIAP, Survivin and Beta-actin.

    Article Snippet: 10–15 µg of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) (Immobilion, Millipore).

    Techniques: Expressing, Electrophoretic Mobility Shift Assay, Lysis, SDS Page, Transfection

    Effect of CSE on HSA Cys34 free sulfhydryl group as determined by the Ellman assay. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% (v/v) CSE and then exhaustively dialyzed. The concentration of Cys34 sulfhydryl groups in HSA samples was determined by the Ellman assay at 412 nm as described under Materials and methods . Data are presented as the mean ± SD of three independent measurements. Inset: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by biotin-HPDP binding and Western blot analysis. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% CSE, exhaustively dialyzed and then labeled at Cys34 with biotin-HPDP as described under Materials and methods . Proteins (10 µg/lane) were separated by SDS-PAGE and biotin-HPDP binding was detected by Western blot analysis using streptavidin-HRP as described under Materials and methods (immunoblot inset). Amido Black staining of the same PVDF membrane showed equal protein loading and transfer (not shown). Immunoblot shown is representative of three independent determinations. Bar-graph inset shows densitometric analysis of biotin-HPDP incorporation. Data are presented as the mean ± SD of three independent determinations.

    Journal: PLoS ONE

    Article Title: Red Blood Cells Protect Albumin from Cigarette Smoke-Induced Oxidation

    doi: 10.1371/journal.pone.0029930

    Figure Lengend Snippet: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by the Ellman assay. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% (v/v) CSE and then exhaustively dialyzed. The concentration of Cys34 sulfhydryl groups in HSA samples was determined by the Ellman assay at 412 nm as described under Materials and methods . Data are presented as the mean ± SD of three independent measurements. Inset: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by biotin-HPDP binding and Western blot analysis. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% CSE, exhaustively dialyzed and then labeled at Cys34 with biotin-HPDP as described under Materials and methods . Proteins (10 µg/lane) were separated by SDS-PAGE and biotin-HPDP binding was detected by Western blot analysis using streptavidin-HRP as described under Materials and methods (immunoblot inset). Amido Black staining of the same PVDF membrane showed equal protein loading and transfer (not shown). Immunoblot shown is representative of three independent determinations. Bar-graph inset shows densitometric analysis of biotin-HPDP incorporation. Data are presented as the mean ± SD of three independent determinations.

    Article Snippet: Samples were then run on SDS–PAGE on Tris–HCl 10% resolving gels and electroblotted on to Immobilon P polyvinylidene difluoride (PVDF; Sigma-Aldrich, Milan, Italy) membrane or stored at −20°C for later use.

    Techniques: Concentration Assay, Binding Assay, Western Blot, Labeling, SDS Page, Staining

    Analysis of oligomerization of Cyt1Aa wild-type and mutant toxins in the presence of SUV. A. Oligomerization of the wild-type Cyt1Aa protein after incubating the solubilized protoxin 1 h at 37°C with proteinase K the presence of SUV liposomes. Samples were heated 3 min at different temperatures before loading into SDS-PAGE. B. Oligomerization of different Cyt1Aa mutants after activation with trypsin or proteinase K in the presence of SUV liposomes. Samples were heated 3 min at 65°C before loaded into SDS-PAGE. C. Analysis of oligomeric structure stability of V122E and V126E mutants after heating at different temperatures. Protoxins were activated with proteinase K in the presence of SUV liposomes, and final samples were heated 3 min at different temperatures before loading into SDS-PAGE. D. Partition of oligomeric structures into the SUV membranes. Oligomerization of the different Cyt1Aa mutants after activation with trypsin or proteinase K in the presence of SUV liposomes. Samples were centrifuged 30 min at 117 000 g . The pellets containing membrane samples and supernatants were separated, heated 3 min at 65°C before SDS-PAGE. All SDS-PAGE gels were transferred to PVDF and analysed by Western blot using polyclonal anti-Cyt1Aa antibody. Molecular weight markers were Precision Plus Protein Standards All Blue (Bio-Rad), and molecular masses are indicated in kDa.

    Journal: Environmental microbiology

    Article Title: Oligomerization is a key step in Cyt1Aa membrane insertion and toxicity but not necessary to synergize Cry11Aa toxicity in Aedes aegypti larvae

    doi: 10.1111/1462-2920.12263

    Figure Lengend Snippet: Analysis of oligomerization of Cyt1Aa wild-type and mutant toxins in the presence of SUV. A. Oligomerization of the wild-type Cyt1Aa protein after incubating the solubilized protoxin 1 h at 37°C with proteinase K the presence of SUV liposomes. Samples were heated 3 min at different temperatures before loading into SDS-PAGE. B. Oligomerization of different Cyt1Aa mutants after activation with trypsin or proteinase K in the presence of SUV liposomes. Samples were heated 3 min at 65°C before loaded into SDS-PAGE. C. Analysis of oligomeric structure stability of V122E and V126E mutants after heating at different temperatures. Protoxins were activated with proteinase K in the presence of SUV liposomes, and final samples were heated 3 min at different temperatures before loading into SDS-PAGE. D. Partition of oligomeric structures into the SUV membranes. Oligomerization of the different Cyt1Aa mutants after activation with trypsin or proteinase K in the presence of SUV liposomes. Samples were centrifuged 30 min at 117 000 g . The pellets containing membrane samples and supernatants were separated, heated 3 min at 65°C before SDS-PAGE. All SDS-PAGE gels were transferred to PVDF and analysed by Western blot using polyclonal anti-Cyt1Aa antibody. Molecular weight markers were Precision Plus Protein Standards All Blue (Bio-Rad), and molecular masses are indicated in kDa.

    Article Snippet: Samples were heated at different temperatures for 3 min, loaded in SDS-PAGE gels and transferred to PVDF Immobilon-P Millipore (Darmstadt, Germany) membranes in a wet chamber during 12 h, 150 mA, at 4°C.

    Techniques: Mutagenesis, SDS Page, Activation Assay, Western Blot, Molecular Weight

    Purification and biochemical characteristics of IPAF. (A) Crude protein extracts were fractionated by a DEAE-52 cellulose column eluted with linear gradient of 0.0–1.0 M NaCl. The elution profile was generated based on protein concentration determined by BCA analysis. (B) The active fractions were further purified using FPLC system equipped with a HiTrapQ anion exchange column eluted with linear gradient of 0.0–1.0 M NaCl. The elution profile was generated by measuring the absorbance at 280 nm. (C) Purified IPAF was identified by SDS-PAGE with Coomassie brilliant blue (lane 2) and periodic acid-Schiff staining (lane 3). Purified IPAF was transferred to PVDF membrane and identified via western blot using a self-made mAb against IPAF (lane 4). The molecular weight of IPAF was determined by comparing with pre-stained protein markers (lane 1). (D) Gel-filtration capillary electrophoresis SDS-MW analysis of IPAF prepared under reducing or non-reducing condition. The MW was derived by normalizing the migration time of the samples with the 10 kDa internal standard, and calibrated with the standard curve constructed with the protein size standard.

    Journal: PLoS ONE

    Article Title: Molecular Cloning of a New Immunomodulatory Protein from Anoectochilus formosanus which Induces B Cell IgM Secretion through a T-Independent Mechanism

    doi: 10.1371/journal.pone.0021004

    Figure Lengend Snippet: Purification and biochemical characteristics of IPAF. (A) Crude protein extracts were fractionated by a DEAE-52 cellulose column eluted with linear gradient of 0.0–1.0 M NaCl. The elution profile was generated based on protein concentration determined by BCA analysis. (B) The active fractions were further purified using FPLC system equipped with a HiTrapQ anion exchange column eluted with linear gradient of 0.0–1.0 M NaCl. The elution profile was generated by measuring the absorbance at 280 nm. (C) Purified IPAF was identified by SDS-PAGE with Coomassie brilliant blue (lane 2) and periodic acid-Schiff staining (lane 3). Purified IPAF was transferred to PVDF membrane and identified via western blot using a self-made mAb against IPAF (lane 4). The molecular weight of IPAF was determined by comparing with pre-stained protein markers (lane 1). (D) Gel-filtration capillary electrophoresis SDS-MW analysis of IPAF prepared under reducing or non-reducing condition. The MW was derived by normalizing the migration time of the samples with the 10 kDa internal standard, and calibrated with the standard curve constructed with the protein size standard.

    Article Snippet: Purified IPAF was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) Immobilon P (Millipore, Billerica, MA) membrane using Trans-Blot Cell system (Bio-Rad) in transfer buffer.

    Techniques: Purification, Generated, Protein Concentration, BIA-KA, Fast Protein Liquid Chromatography, SDS Page, Staining, Western Blot, Molecular Weight, Filtration, Electrophoresis, Derivative Assay, Migration, Construct

    Construction and stable expression of pIgR-ζ chimeras in Jurkat cells. (a) A schematic representation of the pIgR-WT, pIgR-ζ, and pIgRWT-ζ constructs. The extracellular, TMD, and cytoplasmic tails are indicated. Numbers correspond to amino acids after signal peptide cleavage. (b) Stable expression of pIgR-WT, pIgR-ζ, and pIgRWT-ζ in Jurkat cells selected with G418 (2 mg/ml). Cell lysates were immunoprecipitated for pIgR with guinea pig anti-SC conjugated to Protein A-Sepharose, separated by 10% SDS-PAGE, and transferred to immobilon P for detection by Western blot. The pIgR constructs were detected with sheep anti-SC, followed by HRP-labeled secondary antibody. The molecular weight markers in kilodaltons are indicated on the right. Note that each construct migrates as a dimer, due to heterogeneous glycosylation, as previously observed. The bands around 70 kDa are SC, which is cleaved from the pIgR due to the extremely protease-sensitive site where this cleavage normally occurs. (c) Flow cytometry was used to analyze cell surface expression of pIgR-WT, pIgR-ζ, and pIgRWT-ζ. Jurkat cells (1–2 × 10 6 ) were cooled to 4°C and stained with sheep anti-SC antibody followed by anti-sheep FITC-conjugated secondary antibody.

    Journal: Molecular Biology of the Cell

    Article Title: Dimerization of the Polymeric Immunoglobulin Receptor Controls Its Transcytotic Trafficking

    doi:

    Figure Lengend Snippet: Construction and stable expression of pIgR-ζ chimeras in Jurkat cells. (a) A schematic representation of the pIgR-WT, pIgR-ζ, and pIgRWT-ζ constructs. The extracellular, TMD, and cytoplasmic tails are indicated. Numbers correspond to amino acids after signal peptide cleavage. (b) Stable expression of pIgR-WT, pIgR-ζ, and pIgRWT-ζ in Jurkat cells selected with G418 (2 mg/ml). Cell lysates were immunoprecipitated for pIgR with guinea pig anti-SC conjugated to Protein A-Sepharose, separated by 10% SDS-PAGE, and transferred to immobilon P for detection by Western blot. The pIgR constructs were detected with sheep anti-SC, followed by HRP-labeled secondary antibody. The molecular weight markers in kilodaltons are indicated on the right. Note that each construct migrates as a dimer, due to heterogeneous glycosylation, as previously observed. The bands around 70 kDa are SC, which is cleaved from the pIgR due to the extremely protease-sensitive site where this cleavage normally occurs. (c) Flow cytometry was used to analyze cell surface expression of pIgR-WT, pIgR-ζ, and pIgRWT-ζ. Jurkat cells (1–2 × 10 6 ) were cooled to 4°C and stained with sheep anti-SC antibody followed by anti-sheep FITC-conjugated secondary antibody.

    Article Snippet: After the nuclei had been spun out, lysates were cleared 2× with empty protein A Sepharose (Pharmacia, Uppsala, Sweden), immunoprecipitated with anti-ζ monoclonal 6810.2 (a kind gift from Art Weiss) bound to protein A, resolved by 15% SDS-PAGE, and transferred to Immobilon P (Millipore, Bedford, MA) for Western blot with mouse monoclonal 4G10 (anti-phosphotyrosine, Upstate Biotechnology, Lake Placid, NY).

    Techniques: Expressing, Construct, Immunoprecipitation, SDS Page, Western Blot, Labeling, Molecular Weight, Flow Cytometry, Cytometry, Staining

    Western blotting confirms the presence of ApoE and Na + /K + -ATPase α-chains in SAF preparations. Based on an estimate of 10 µg PrP Sc per brain, the equivalent of 0.25 µg/µl PrP Sc from an ME7 (lane 1), 22F (lane 2) and 79A (lane 3) SAF preparation were resolved by SDS-PAGE, blotted onto PVDF membrane and probed with the primary antibody against (A) Apolipoprotein E (B) Total Na + /K + ATPase α-chains using a pan α-chain antibody (C) Na + /K + ATPase α2 isoform and (D) Na + /K + ATPase α3 isoform. In all cases, in lanes 4 and 5 were loaded the equivalent of 0.25 µg/µl of control preparation material from uninfected WT and PrP −/− mouse brains respectively. Molecular weight markers are in kDa.

    Journal: PLoS ONE

    Article Title: Na+/K+-ATPase Is Present in Scrapie-Associated Fibrils, Modulates PrP Misfolding In Vitro and Links PrP Function and Dysfunction

    doi: 10.1371/journal.pone.0026813

    Figure Lengend Snippet: Western blotting confirms the presence of ApoE and Na + /K + -ATPase α-chains in SAF preparations. Based on an estimate of 10 µg PrP Sc per brain, the equivalent of 0.25 µg/µl PrP Sc from an ME7 (lane 1), 22F (lane 2) and 79A (lane 3) SAF preparation were resolved by SDS-PAGE, blotted onto PVDF membrane and probed with the primary antibody against (A) Apolipoprotein E (B) Total Na + /K + ATPase α-chains using a pan α-chain antibody (C) Na + /K + ATPase α2 isoform and (D) Na + /K + ATPase α3 isoform. In all cases, in lanes 4 and 5 were loaded the equivalent of 0.25 µg/µl of control preparation material from uninfected WT and PrP −/− mouse brains respectively. Molecular weight markers are in kDa.

    Article Snippet: For Western blotting, gels were semi-dry blotted onto Immobilon-P PVDF membrane (Whatman).

    Techniques: Western Blot, SDS Page, Molecular Weight

    In vitro cleavage of WT vSag7 or the CS12X triple mutant by cathepsin L. Equal amounts of purified [ 35 S]methionine-labeled proteins produced with a coupled in vitro transcription-translation system were subjected to cleavage with 10 ng of purified cathepsin L, fractionated by SDS–15% PAGE, transferred to PVDF membranes, and exposed to a PhosphorImager screen. Time of cleavage is in minutes. These data are representative of three independent experiments. The values on the left are the molecular masses in kilodaltons.

    Journal: Journal of Virology

    Article Title: Alternative Proteolytic Processing of Mouse Mammary Tumor Virus Superantigens

    doi:

    Figure Lengend Snippet: In vitro cleavage of WT vSag7 or the CS12X triple mutant by cathepsin L. Equal amounts of purified [ 35 S]methionine-labeled proteins produced with a coupled in vitro transcription-translation system were subjected to cleavage with 10 ng of purified cathepsin L, fractionated by SDS–15% PAGE, transferred to PVDF membranes, and exposed to a PhosphorImager screen. Time of cleavage is in minutes. These data are representative of three independent experiments. The values on the left are the molecular masses in kilodaltons.

    Article Snippet: Products were fractionated by sodium dodecyl sulfate (SDS)–15% polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membranes (Roche Diagnostics), and exposed overnight on PhosphorImager screens (Molecular Dynamics, Sunnyvale, Calif.).

    Techniques: In Vitro, Mutagenesis, Purification, Labeling, Produced, Polyacrylamide Gel Electrophoresis

    In vitro cleavage of vSag7 mutants by recombinant convertases. vSags were produced with a coupled in vitro transcription-and-translation system as [ 35 S]methionine-labeled proteins. Equal amounts of Ni-nitrilotriacetic acid-purified material were subjected to cleavage with equal units of convertase activity. The mutated and WT vSags were digested overnight. Cleavage products were separated by SDS–15% PAGE, transferred to PVDF membranes, and exposed on PhosphorImager screens. (A) Expected molecular weights of cleavage products. (B) Cleavage of the CS2X mutant. (C) Cleavage of the CS1X mutant. (D) Cleavage of WT vSag7. (E) Cleavage of the CS12 mutant. Lanes: 1, furin; 2, PC5; 3, PC7; 4, furin plus EDTA; 5, uncleaved control. These data are representative of three independent experiments. The values shown are the molecular masses in kilodaltons.

    Journal: Journal of Virology

    Article Title: Alternative Proteolytic Processing of Mouse Mammary Tumor Virus Superantigens

    doi:

    Figure Lengend Snippet: In vitro cleavage of vSag7 mutants by recombinant convertases. vSags were produced with a coupled in vitro transcription-and-translation system as [ 35 S]methionine-labeled proteins. Equal amounts of Ni-nitrilotriacetic acid-purified material were subjected to cleavage with equal units of convertase activity. The mutated and WT vSags were digested overnight. Cleavage products were separated by SDS–15% PAGE, transferred to PVDF membranes, and exposed on PhosphorImager screens. (A) Expected molecular weights of cleavage products. (B) Cleavage of the CS2X mutant. (C) Cleavage of the CS1X mutant. (D) Cleavage of WT vSag7. (E) Cleavage of the CS12 mutant. Lanes: 1, furin; 2, PC5; 3, PC7; 4, furin plus EDTA; 5, uncleaved control. These data are representative of three independent experiments. The values shown are the molecular masses in kilodaltons.

    Article Snippet: Products were fractionated by sodium dodecyl sulfate (SDS)–15% polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membranes (Roche Diagnostics), and exposed overnight on PhosphorImager screens (Molecular Dynamics, Sunnyvale, Calif.).

    Techniques: In Vitro, Recombinant, Produced, Labeling, Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Mutagenesis

    The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to PVDF membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. Immunoblot of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).

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

    Article Title: Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport

    doi: 10.1073/pnas.1719070115

    Figure Lengend Snippet: The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to PVDF membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. Immunoblot of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).

    Article Snippet: For immunoblot analyses, proteins were transferred to polyvinylidene fluoride (PVDF) membrane by semidry electroblotting in Trans-Blot Turbo Transfer System (BioRad).

    Techniques: Isolation, SDS Page, Staining, Immunodetection

    Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.

    Article Snippet: SDS–PAGE and Western blotting Samples were separated by SDS–PAGE on 4-12% Bis-Tris polyacrylamide gels (Life Technologies) using PAGE Ruler (Thermo Fisher Scientific) as a size marker and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA).

    Techniques: Cell Culture, SDS Page

    Calpain cleaves otoferlin and myoferlin in addition to dysferlin. (A) Calpain rapidly cleaves immunoprecipitated ferlin proteins in vitro. Dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag were immunoprecipitated with anti-myc and protein G–Sepharose (see Materials and Methods ). Dysferlin-bound Sepharose beads were incubated with purified 0.2 A.U. of recombinant calpain-1 at 30°C for 2 or 10 s in the presence of 2 mM CaCl 2. Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94°C. Digested samples were analyzed by SDS–PAGE and Western blot. Top, C-terminal fragments detected with anti-myc (dysferlin) or anti-Flag (myoferlin and otoferlin). Bottom, N-terminal fragments detected by N-terminal (Romeo-dysferlin) or internal antibodies (7D2, myoferlin; C12, otoferlin). (B) Dysferlin and otoferlin display damage-dependent cleavage, whereas myoferlin cleavage appears to be constitutive. HEK293 cells were transfected with dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag and lysed in calcium-free RIPA (lane 1), RIPA containing 900 μM calcium (permissive for calpain cleavage), or damaged by scraping in the presence of calcium. Scraped cell pellets were lysed in calcium-free RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. Dysferlin was detected with anti-Myc; otoferlin and myoferlin were detected with anti-Flag. (C) Diagram of the predicted calpain cleavage sites within dysferlin, otoferlin, and myoferlin (schematic produced using DOG 2.0; Ren et al. , 2009 ). Molecular weight calculation of the cleaved C-terminal modules was used to elucidate the most likely calpain cleavage site ( ccd.biocuckoo.org ). In each case, the C-terminal fragments released by calpain cleavage represent transmembrane-anchored, dual-C2-domain modules.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Calpain cleaves otoferlin and myoferlin in addition to dysferlin. (A) Calpain rapidly cleaves immunoprecipitated ferlin proteins in vitro. Dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag were immunoprecipitated with anti-myc and protein G–Sepharose (see Materials and Methods ). Dysferlin-bound Sepharose beads were incubated with purified 0.2 A.U. of recombinant calpain-1 at 30°C for 2 or 10 s in the presence of 2 mM CaCl 2. Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94°C. Digested samples were analyzed by SDS–PAGE and Western blot. Top, C-terminal fragments detected with anti-myc (dysferlin) or anti-Flag (myoferlin and otoferlin). Bottom, N-terminal fragments detected by N-terminal (Romeo-dysferlin) or internal antibodies (7D2, myoferlin; C12, otoferlin). (B) Dysferlin and otoferlin display damage-dependent cleavage, whereas myoferlin cleavage appears to be constitutive. HEK293 cells were transfected with dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag and lysed in calcium-free RIPA (lane 1), RIPA containing 900 μM calcium (permissive for calpain cleavage), or damaged by scraping in the presence of calcium. Scraped cell pellets were lysed in calcium-free RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. Dysferlin was detected with anti-Myc; otoferlin and myoferlin were detected with anti-Flag. (C) Diagram of the predicted calpain cleavage sites within dysferlin, otoferlin, and myoferlin (schematic produced using DOG 2.0; Ren et al. , 2009 ). Molecular weight calculation of the cleaved C-terminal modules was used to elucidate the most likely calpain cleavage site ( ccd.biocuckoo.org ). In each case, the C-terminal fragments released by calpain cleavage represent transmembrane-anchored, dual-C2-domain modules.

    Article Snippet: SDS–PAGE and Western blotting Samples were separated by SDS–PAGE on 4-12% Bis-Tris polyacrylamide gels (Life Technologies) using PAGE Ruler (Thermo Fisher Scientific) as a size marker and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA).

    Techniques: Immunoprecipitation, In Vitro, Incubation, Purification, Recombinant, Lysis, SDS Page, Western Blot, Transfection, Produced, Molecular Weight

    Immunoblot analysis of r-OspF protein family members expressed in E. coli . Each member of the ospF gene family was cloned and expressed as an S-tag fusion protein using ligase-independent cloning methods as described in the text. Proteins from E. coli cultures that were induced to express the r-proteins with IPTG were fractionated by SDS-PAGE and transferred to a PVDF membrane by electroblotting. The membrane on the left was screened with anti-S-Tag protein HRP conjugate, while the membrane on the right was screened with a polyclonal anti-OspF antiserum (generated with a gene of B. burgdorferi N40 origin). Immunoblot methods are described in the text. Molecular size standards are indicated on the left.

    Journal: Infection and Immunity

    Article Title: Demonstration of the Genetic Stability and Temporal Expression of Select Members of the Lyme Disease Spirochete OspF Protein Family during Infection in Mice

    doi: 10.1128/IAI.69.8.4831-4838.2001

    Figure Lengend Snippet: Immunoblot analysis of r-OspF protein family members expressed in E. coli . Each member of the ospF gene family was cloned and expressed as an S-tag fusion protein using ligase-independent cloning methods as described in the text. Proteins from E. coli cultures that were induced to express the r-proteins with IPTG were fractionated by SDS-PAGE and transferred to a PVDF membrane by electroblotting. The membrane on the left was screened with anti-S-Tag protein HRP conjugate, while the membrane on the right was screened with a polyclonal anti-OspF antiserum (generated with a gene of B. burgdorferi N40 origin). Immunoblot methods are described in the text. Molecular size standards are indicated on the left.

    Article Snippet: To conduct immunoblot analyses, the proteins were transferred from the gels onto polyvinylidene difluoride (PVDF) by electroblotting using the Trans-blot system (Bio-Rad) as previously described ( ).

    Techniques: Clone Assay, Ligase Independent Cloning, SDS Page, Generated

    EndoS-hydrolyzed IgG dominantly inhibit inflammation. ( A ) SDS/PAGE and lectin blot analysis of mAbs incubated with (+) or without (−) EndoS hydrolysis and separated by 10% SDS/PAGE. The proteins were detected by PageBlue stain (Stain) or by blotting onto a PVDF membrane probed with Lens culinaris agglutinin (LCA). ( B ) Representative figures of H E-stained ankle joints of mice ( n = 3–4 per group) injected with anti-CII mAbs; unhydrolyzed ( Left ), EndoS-hydrolyzed ( Center ), or mixed IgG ( Right ). Magnification ×10. ( C ) Hy2.15 and ( D ) EndoS-treated Hy2.15. Shown spectra were acquired during the time period for which the majority of glycosylated peptides from EEQFNSTFR (21.5–23.0 min) elute. Doubly and triply charged ions as well as predicted glycan structures are shown. All numbers given are for the monoisotopic mass charge. In all of the animal experiments, male ( BALB/c × B10.Q) F1 mice were used. Unless otherwise stated all of the mice received antibodies i.v. (d 0) and 25 μg of LPS i.p. (d 5). For arthritis induction in experiments shown in E , F , I and J , 9 mg of two anti-CII mAb mixtures (M2139 + CIIC1) were used, whereas for experiments in G and H 4 mg of four anti-CII mAb mixture (M2139 + CIIC1 + CIIC2 + UL1) was used. Antigen specificity is not required for inhibition. Mice ( n = 42) were injected with 4 mg of EndoS-hydrolyzed IgG ( E ) M2139H + CIIC1H or UL1H + CIIC2H or ( F ) Hy2.15H + L243H followed by anti-CII mAb. Dose and subclass dependency. ( G ) Mice ( n = 39) were injected with EndoS-hydrolyzed or unhydrolyzed IgG1 (Hy2.15) or IgG2a (L243) mAb binding to joint unrelated antigens at two different concentrations (1 mg and 0.25 mg), followed by anti-CII mAb. ( H ) Mice ( n = 65) were injected with different subclasses of EndoS-hydrolyzed anti-CII (M284H, M2139H, CIIC1H, CIIC2H, and UL1H) or anti-citrullinated CII peptide IgG (ACC4H) at two different concentrations (1 mg and 0.25 mg), followed by anti-CII mAb. ( I ) Mice ( n = 25) were injected with a mixture of EndoS-hydrolyzed and/or unhydrolyzed anti-CII IgG at different combinations. In mixed IgG groups, group 1 received 4.5 mg of unhydrolyzed and 4.5 mg of EndoS-hydrolyzed IgG, group 2 had 6.75 mg of unhydrolyzed and 2.25 mg of EndoS-hydrolyzed IgG, and group 3 received 7.8775 mg of unhydrolyzed and 1.125 mg of EndoS-hydrolyzed IgG. ( J ) Mice ( n = 25) were injected with different concentrations (50–4,000 μg) of EndoS-hydrolyzed single anti-CII IgG (M2139H), followed by anti-CII mAb. Three hours after the antibody transfer, LPS was injected. H denotes EndoS-hydrolyzed IgG. Hy2.15 and L243 represent mAbs binding to TNP hapten and human HLA-DR antigen, respectively. Error bars indicate ± SEM.

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

    Article Title: From the Cover: Dominant suppression of inflammation by glycan-hydrolyzed IgG

    doi: 10.1073/pnas.1301480110

    Figure Lengend Snippet: EndoS-hydrolyzed IgG dominantly inhibit inflammation. ( A ) SDS/PAGE and lectin blot analysis of mAbs incubated with (+) or without (−) EndoS hydrolysis and separated by 10% SDS/PAGE. The proteins were detected by PageBlue stain (Stain) or by blotting onto a PVDF membrane probed with Lens culinaris agglutinin (LCA). ( B ) Representative figures of H E-stained ankle joints of mice ( n = 3–4 per group) injected with anti-CII mAbs; unhydrolyzed ( Left ), EndoS-hydrolyzed ( Center ), or mixed IgG ( Right ). Magnification ×10. ( C ) Hy2.15 and ( D ) EndoS-treated Hy2.15. Shown spectra were acquired during the time period for which the majority of glycosylated peptides from EEQFNSTFR (21.5–23.0 min) elute. Doubly and triply charged ions as well as predicted glycan structures are shown. All numbers given are for the monoisotopic mass charge. In all of the animal experiments, male ( BALB/c × B10.Q) F1 mice were used. Unless otherwise stated all of the mice received antibodies i.v. (d 0) and 25 μg of LPS i.p. (d 5). For arthritis induction in experiments shown in E , F , I and J , 9 mg of two anti-CII mAb mixtures (M2139 + CIIC1) were used, whereas for experiments in G and H 4 mg of four anti-CII mAb mixture (M2139 + CIIC1 + CIIC2 + UL1) was used. Antigen specificity is not required for inhibition. Mice ( n = 42) were injected with 4 mg of EndoS-hydrolyzed IgG ( E ) M2139H + CIIC1H or UL1H + CIIC2H or ( F ) Hy2.15H + L243H followed by anti-CII mAb. Dose and subclass dependency. ( G ) Mice ( n = 39) were injected with EndoS-hydrolyzed or unhydrolyzed IgG1 (Hy2.15) or IgG2a (L243) mAb binding to joint unrelated antigens at two different concentrations (1 mg and 0.25 mg), followed by anti-CII mAb. ( H ) Mice ( n = 65) were injected with different subclasses of EndoS-hydrolyzed anti-CII (M284H, M2139H, CIIC1H, CIIC2H, and UL1H) or anti-citrullinated CII peptide IgG (ACC4H) at two different concentrations (1 mg and 0.25 mg), followed by anti-CII mAb. ( I ) Mice ( n = 25) were injected with a mixture of EndoS-hydrolyzed and/or unhydrolyzed anti-CII IgG at different combinations. In mixed IgG groups, group 1 received 4.5 mg of unhydrolyzed and 4.5 mg of EndoS-hydrolyzed IgG, group 2 had 6.75 mg of unhydrolyzed and 2.25 mg of EndoS-hydrolyzed IgG, and group 3 received 7.8775 mg of unhydrolyzed and 1.125 mg of EndoS-hydrolyzed IgG. ( J ) Mice ( n = 25) were injected with different concentrations (50–4,000 μg) of EndoS-hydrolyzed single anti-CII IgG (M2139H), followed by anti-CII mAb. Three hours after the antibody transfer, LPS was injected. H denotes EndoS-hydrolyzed IgG. Hy2.15 and L243 represent mAbs binding to TNP hapten and human HLA-DR antigen, respectively. Error bars indicate ± SEM.

    Article Snippet: Briefly, 2 µg of EndoS-hydrolyzed and unhydrolyzed IgG were separated on 10% SDS/PAGE followed by staining with PageBlue protein stain (ThermoFisher Scientific), or blotted to PVDF using TransBlot Turbo transfer packs and apparatus (Bio-Rad).

    Techniques: SDS Page, Incubation, Staining, Mouse Assay, Injection, Inhibition, Binding Assay