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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
    https://www.bioz.com/result/polyvinylidene difluoride pvdf membranes/product/Thermo Fisher
    Average 99 stars, based on 2211 article reviews
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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 16335 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
    https://www.bioz.com/result/immobilon p pvdf membrane/product/GE Healthcare
    Average 92 stars, based on 24 article reviews
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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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    Average 93 stars, based on 629 article reviews
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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
    https://www.bioz.com/result/polyvinylidene difluoride pvdf membrane/product/PerkinElmer
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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
    https://www.bioz.com/result/polyvinylidene fluoride pvdf membrane/product/Merck KGaA
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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
    https://www.bioz.com/result/polyvinylidene fluoride pvdf membrane/product/Bio-Rad
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    91
    Merck & Co polyvinylidene fluoride pvdf membranes
    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 Membranes, supplied by Merck & Co, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyvinylidene fluoride pvdf membranes/product/Merck & Co
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    88
    Fisher Scientific immobilon polyvinyldifluoride pvdf membranes
    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 ).
    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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    99
    Millipore immobilon p pvdf blotting membrane
    The age-associated decrease in BiP specific activity correlates with increased carbonylation. (A) BiP purified from young and aged mouse liver was analyzed for carbonyl content by Western blot analysis. Protein samples (~100 ng) from each age group were derivatized with 2,4-dinitrophenylhydrazine, as described in Materials and methods, resolved on SDS-PAGE and transferred to a <t>PVDF</t> membrane. Relative levels of carbonylation were compared by probing blots with an anti-DNP antibody; the Coomassie Blue stained membrane was used to normalize protein loading. Densitometric analysis was conducted for determination of fold change in carbonylation of BiP with age. (B) The ATPase activity of BiP was quantified using hydrolysis of [γ- 32 P] ATP as described in Materials and methods. Average activity values were calculated from three independent experiments and error bars represent calculated standard errors of mean.
    Immobilon P Pvdf Blotting Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 59 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

    SP-A does not influence cell-surface expression of EGFR in A549 cells. A549 cells were serum-starved overnight. The next day, cells were incubated with 20 μg/ml SP-A for 2 h, washed, and incubated with 0.5 mg/ml Sulfo-NHS-LC-biotin for 30 min at 4 °C. Whole-cell lysates were immunoprecipitated with the monoclonal anti-EGFR antibody (clone Ab-11) or control IgG. Samples were separated by SDS-PAGE, transferred onto PVDF membranes, and probed with HRP-conjugated streptavidin ( right panel ) or the monoclonal anti-human EGFR antibody (clone D38B1), and the bands were detected using HRP-conjugated anti-rabbit IgG ( left panel ). The data are representative of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Surfactant protein A down-regulates epidermal growth factor receptor by mechanisms different from those of surfactant protein D

    doi: 10.1074/jbc.M117.800771

    Figure Lengend Snippet: SP-A does not influence cell-surface expression of EGFR in A549 cells. A549 cells were serum-starved overnight. The next day, cells were incubated with 20 μg/ml SP-A for 2 h, washed, and incubated with 0.5 mg/ml Sulfo-NHS-LC-biotin for 30 min at 4 °C. Whole-cell lysates were immunoprecipitated with the monoclonal anti-EGFR antibody (clone Ab-11) or control IgG. Samples were separated by SDS-PAGE, transferred onto PVDF membranes, and probed with HRP-conjugated streptavidin ( right panel ) or the monoclonal anti-human EGFR antibody (clone D38B1), and the bands were detected using HRP-conjugated anti-rabbit IgG ( left panel ). The data are representative of three independent experiments.

    Article Snippet: The immunoprecipitates were separated by SDS-PAGE and transferred onto PVDF membranes.

    Techniques: Expressing, Incubation, Immunoprecipitation, SDS Page

    SP-A binds to EGFR in A549 cells, H441 cells, and CHOK1 cells stably expressing EGFR. A, whole-cell lysate of A549 cells was immunoprecipitated ( IP ) with anti-EGFR monoclonal antibody Ab-11 or control IgG (0. 8 μg) at 4 °C for 16 h. The samples and BSA (200 ng/lane) were subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with or without SP-A (1 μg/ml) for 16 h. The membranes not incubated with SP-A were subjected to Western blotting using an anti-human EGFR monoclonal antibody ( WB: EGFR ). The membranes incubated with SP-A were then treated with an anti-human SP-A polyclonal antibody ( SP-A pAb ) or monoclonal antibodies PE10 ( SP-A mAb (PE10 )) or PC6 ( SP-A mAb (PC6 )), which was followed by incubation with HRP-labeled anti-rabbit IgG or anti-mouse IgG ( upper panel ). As a negative control, the membranes in the absence of incubation with SP-A were also incubated with an anti-human SP-A polyclonal antibody ( WB: SP-A pAb ) or monoclonal antibodies PE10 ( WB: SP-A mAb (PE10 )) or PC6 ( WB: SP-A mAb (PC6 )), which was followed by incubation with HRP-labeled anti-rabbit IgG or anti-mouse IgG ( lower panel ). The data are representative of three independent experiments. B and C, experimental paradigm described in A was performed in H441 cells ( B ) and CHOK1 cells stably expressing EGFR ( C ) by using an anti-human SP-A polyclonal antibody, which was followed by incubation with HRP-labeled anti-rabbit IgG. The data are representative of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Surfactant protein A down-regulates epidermal growth factor receptor by mechanisms different from those of surfactant protein D

    doi: 10.1074/jbc.M117.800771

    Figure Lengend Snippet: SP-A binds to EGFR in A549 cells, H441 cells, and CHOK1 cells stably expressing EGFR. A, whole-cell lysate of A549 cells was immunoprecipitated ( IP ) with anti-EGFR monoclonal antibody Ab-11 or control IgG (0. 8 μg) at 4 °C for 16 h. The samples and BSA (200 ng/lane) were subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with or without SP-A (1 μg/ml) for 16 h. The membranes not incubated with SP-A were subjected to Western blotting using an anti-human EGFR monoclonal antibody ( WB: EGFR ). The membranes incubated with SP-A were then treated with an anti-human SP-A polyclonal antibody ( SP-A pAb ) or monoclonal antibodies PE10 ( SP-A mAb (PE10 )) or PC6 ( SP-A mAb (PC6 )), which was followed by incubation with HRP-labeled anti-rabbit IgG or anti-mouse IgG ( upper panel ). As a negative control, the membranes in the absence of incubation with SP-A were also incubated with an anti-human SP-A polyclonal antibody ( WB: SP-A pAb ) or monoclonal antibodies PE10 ( WB: SP-A mAb (PE10 )) or PC6 ( WB: SP-A mAb (PC6 )), which was followed by incubation with HRP-labeled anti-rabbit IgG or anti-mouse IgG ( lower panel ). The data are representative of three independent experiments. B and C, experimental paradigm described in A was performed in H441 cells ( B ) and CHOK1 cells stably expressing EGFR ( C ) by using an anti-human SP-A polyclonal antibody, which was followed by incubation with HRP-labeled anti-rabbit IgG. The data are representative of three independent experiments.

    Article Snippet: The immunoprecipitates were separated by SDS-PAGE and transferred onto PVDF membranes.

    Techniques: Stable Transfection, Expressing, Immunoprecipitation, SDS Page, Incubation, Western Blot, Labeling, Negative Control

    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

    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

    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

    The age-associated decrease in BiP specific activity correlates with increased carbonylation. (A) BiP purified from young and aged mouse liver was analyzed for carbonyl content by Western blot analysis. Protein samples (~100 ng) from each age group were derivatized with 2,4-dinitrophenylhydrazine, as described in Materials and methods, resolved on SDS-PAGE and transferred to a PVDF membrane. Relative levels of carbonylation were compared by probing blots with an anti-DNP antibody; the Coomassie Blue stained membrane was used to normalize protein loading. Densitometric analysis was conducted for determination of fold change in carbonylation of BiP with age. (B) The ATPase activity of BiP was quantified using hydrolysis of [γ- 32 P] ATP as described in Materials and methods. Average activity values were calculated from three independent experiments and error bars represent calculated standard errors of mean.

    Journal: Biochemical and biophysical research communications

    Article Title: DECREASED ENZYME ACTIVITIES OF CHAPERONES PDI AND BIP IN AGED MOUSE LIVERS

    doi: 10.1016/j.bbrc.2007.10.194

    Figure Lengend Snippet: The age-associated decrease in BiP specific activity correlates with increased carbonylation. (A) BiP purified from young and aged mouse liver was analyzed for carbonyl content by Western blot analysis. Protein samples (~100 ng) from each age group were derivatized with 2,4-dinitrophenylhydrazine, as described in Materials and methods, resolved on SDS-PAGE and transferred to a PVDF membrane. Relative levels of carbonylation were compared by probing blots with an anti-DNP antibody; the Coomassie Blue stained membrane was used to normalize protein loading. Densitometric analysis was conducted for determination of fold change in carbonylation of BiP with age. (B) The ATPase activity of BiP was quantified using hydrolysis of [γ- 32 P] ATP as described in Materials and methods. Average activity values were calculated from three independent experiments and error bars represent calculated standard errors of mean.

    Article Snippet: Fractions containing PDI and BiP were identified by resolving a small aliquot of these fractions on large format 26 well acrylamide gels (Bio-Rad), transferring the gels to Immobilon-P (Millipore) PVDF membranes and probing membranes with antibodies specific for either PDI or BiP.

    Techniques: Activity Assay, Purification, Western Blot, SDS Page, Staining

    The age-associated decrease in PDI specific activity correlates with increased carbonylation. (A) PDI purified from young and aged mouse livers was analyzed for carbonyl content by Western blot analysis. Protein samples (~200 ng) from each age group were derivatized with 2,4-dinitrophenylhydrazine, as described in Materials and methods, resolved on SDS-PAGE and transferred to a PVDF membrane. Relative levels of carbonylation were compared by probing blots with an anti-DNP antibody; the Coomassie Blue stained membrane was used to normalize protein loading. Densitometric analysis was conducted on the band present in both young and aged samples (indicated with arrow on the figure). (B) Specific activity for young and aged PDI was measured as GSH-dependent reduction of insulin as described in Materials and methods. The average values reported for each age group were obtained from five independent experiments and error bars represent calculated standard errors of mean.

    Journal: Biochemical and biophysical research communications

    Article Title: DECREASED ENZYME ACTIVITIES OF CHAPERONES PDI AND BIP IN AGED MOUSE LIVERS

    doi: 10.1016/j.bbrc.2007.10.194

    Figure Lengend Snippet: The age-associated decrease in PDI specific activity correlates with increased carbonylation. (A) PDI purified from young and aged mouse livers was analyzed for carbonyl content by Western blot analysis. Protein samples (~200 ng) from each age group were derivatized with 2,4-dinitrophenylhydrazine, as described in Materials and methods, resolved on SDS-PAGE and transferred to a PVDF membrane. Relative levels of carbonylation were compared by probing blots with an anti-DNP antibody; the Coomassie Blue stained membrane was used to normalize protein loading. Densitometric analysis was conducted on the band present in both young and aged samples (indicated with arrow on the figure). (B) Specific activity for young and aged PDI was measured as GSH-dependent reduction of insulin as described in Materials and methods. The average values reported for each age group were obtained from five independent experiments and error bars represent calculated standard errors of mean.

    Article Snippet: Fractions containing PDI and BiP were identified by resolving a small aliquot of these fractions on large format 26 well acrylamide gels (Bio-Rad), transferring the gels to Immobilon-P (Millipore) PVDF membranes and probing membranes with antibodies specific for either PDI or BiP.

    Techniques: Activity Assay, Purification, Western Blot, SDS Page, Staining

    Western blotting analysis of Halobacterium sp. NRC-1 andderivative strains using rabbit Rfa3 antiserum. A. Equal quantities of totalcell lysate protein samples were electrophoresed on a 12% polyacrylamide-SDSgel, transferred to PVDF membrane, and probed

    Journal: Applied microbiology and biotechnology

    Article Title: Bioengineering radioresistance by overproduction of RPA, a mammalian-type single-stranded DNA-binding protein, in a halophilic archaeon

    doi: 10.1007/s00253-013-5368-x

    Figure Lengend Snippet: Western blotting analysis of Halobacterium sp. NRC-1 andderivative strains using rabbit Rfa3 antiserum. A. Equal quantities of totalcell lysate protein samples were electrophoresed on a 12% polyacrylamide-SDSgel, transferred to PVDF membrane, and probed

    Article Snippet: Briefly, the electrophoretically fractionated cell lysates orpure protein were electroblotted onto 0.45 μm Immobilon-P polyvinylidenedifluoride (PVDF) membranes (Millipore Corp., Boston, MA) for 1 h at 100 voltsusing a Bio-Rad mini gel blotter.

    Techniques: Western Blot

    Oligomerization of Cyt1Aa and mutant proteins after activation of solubilized protoxin, with trypsin in the presence of SUV liposomes. Samples were heated 3 min at 65 °C before loading into the SDS-PAGE transferred to PVDF and reveled in western blot assay as described in materials and methods using polyclonal anti-Cyt1A antibody and goat anti-rabbit antibody coupled to horseradish peroxidase.

    Journal: Scientific Reports

    Article Title: Susceptible and mCry3A resistant corn rootworm larvae killed by a non-hemolytic Bacillus thuringiensis Cyt1Aa mutant

    doi: 10.1038/s41598-018-36205-6

    Figure Lengend Snippet: Oligomerization of Cyt1Aa and mutant proteins after activation of solubilized protoxin, with trypsin in the presence of SUV liposomes. Samples were heated 3 min at 65 °C before loading into the SDS-PAGE transferred to PVDF and reveled in western blot assay as described in materials and methods using polyclonal anti-Cyt1A antibody and goat anti-rabbit antibody coupled to horseradish peroxidase.

    Article Snippet: Proteins were transferred to PVDF Immobilon-P Millipore membranes using a wet chamber (12 h, 150 mA, 4 °C) and revealed by western blot assay as described , using 5% skimmed milk in PBS for blocking (1 h, room temperature), and two washes (5 min each) with PBS containing 0.1% Tween 20 (PBS-Tween).

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