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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.
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1) Product Images from "Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair"

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

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E14-04-0947

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

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

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Far Western Blot:

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Western Blot:

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Marker:

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Staining:

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SDS Page:

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    Merck KGaA immobilon p membrane
    Short EPIYA-phosphopeptides of H. pylori CagA are sufficient for detection by α-phosphotyrosine antibodies. ( A ) Typical Western CagA proteins of H. pylori such as that of strain 26695 [76] contain the EPIYA-A, EPIYA-B, and EPIYA-C segments as indicated. These motifs represent tyrosine phosphorylation sites, which can be phosphorylated by c-Abl and c-Src host kinases. ( B ) The indicated phospho- and non-phospho peptides of the EPIYA-A motif were synthesized and immobilized on <t>PVDF</t> membranes using a Dotblot apparatus. All Dotblots were probed with the indicated commercial phosphotyrosine antibodies and exposed as described in the Material Methods section. Quantified spot intensities of the Dotblots from three independent experiments are shown to the right. Signal intensities were measured densitometrically with the Lumi-Imager F1 and revealed the percentage of phosphorylation signal per sample. The strongest spot on every Dotblot was set at 100% for each of the different α-phosphotyrosine antibodies as indicated. The results show that 11-mer and 9-mer phosphopeptides are sufficient for strong recognition by the antibodies. ( C ) Control Dotblot analyses used products of in vitro kinase reactions of c-Abl with either bacterial lysates (from H. pylori wild-type strain 26695 and isogenic Δ cagA mutant) or a purified recombinant CagA C-terminal fragment. Phosphorylated CagA proteins can be also detected by this Dotblot method using seven phosphotyrosine antibodies ( Table 1 ), while the non-phosphorylated CagA forms cannot.
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    Short EPIYA-phosphopeptides of H. pylori CagA are sufficient for detection by α-phosphotyrosine antibodies. ( A ) Typical Western CagA proteins of H. pylori such as that of strain 26695 [76] contain the EPIYA-A, EPIYA-B, and EPIYA-C segments as indicated. These motifs represent tyrosine phosphorylation sites, which can be phosphorylated by c-Abl and c-Src host kinases. ( B ) The indicated phospho- and non-phospho peptides of the EPIYA-A motif were synthesized and immobilized on PVDF membranes using a Dotblot apparatus. All Dotblots were probed with the indicated commercial phosphotyrosine antibodies and exposed as described in the Material Methods section. Quantified spot intensities of the Dotblots from three independent experiments are shown to the right. Signal intensities were measured densitometrically with the Lumi-Imager F1 and revealed the percentage of phosphorylation signal per sample. The strongest spot on every Dotblot was set at 100% for each of the different α-phosphotyrosine antibodies as indicated. The results show that 11-mer and 9-mer phosphopeptides are sufficient for strong recognition by the antibodies. ( C ) Control Dotblot analyses used products of in vitro kinase reactions of c-Abl with either bacterial lysates (from H. pylori wild-type strain 26695 and isogenic Δ cagA mutant) or a purified recombinant CagA C-terminal fragment. Phosphorylated CagA proteins can be also detected by this Dotblot method using seven phosphotyrosine antibodies ( Table 1 ), while the non-phosphorylated CagA forms cannot.

    Journal: PLoS ONE

    Article Title: Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains

    doi: 10.1371/journal.pone.0096488

    Figure Lengend Snippet: Short EPIYA-phosphopeptides of H. pylori CagA are sufficient for detection by α-phosphotyrosine antibodies. ( A ) Typical Western CagA proteins of H. pylori such as that of strain 26695 [76] contain the EPIYA-A, EPIYA-B, and EPIYA-C segments as indicated. These motifs represent tyrosine phosphorylation sites, which can be phosphorylated by c-Abl and c-Src host kinases. ( B ) The indicated phospho- and non-phospho peptides of the EPIYA-A motif were synthesized and immobilized on PVDF membranes using a Dotblot apparatus. All Dotblots were probed with the indicated commercial phosphotyrosine antibodies and exposed as described in the Material Methods section. Quantified spot intensities of the Dotblots from three independent experiments are shown to the right. Signal intensities were measured densitometrically with the Lumi-Imager F1 and revealed the percentage of phosphorylation signal per sample. The strongest spot on every Dotblot was set at 100% for each of the different α-phosphotyrosine antibodies as indicated. The results show that 11-mer and 9-mer phosphopeptides are sufficient for strong recognition by the antibodies. ( C ) Control Dotblot analyses used products of in vitro kinase reactions of c-Abl with either bacterial lysates (from H. pylori wild-type strain 26695 and isogenic Δ cagA mutant) or a purified recombinant CagA C-terminal fragment. Phosphorylated CagA proteins can be also detected by this Dotblot method using seven phosphotyrosine antibodies ( Table 1 ), while the non-phosphorylated CagA forms cannot.

    Article Snippet: These peptide samples were spotted onto Immobilon-P membrane (Merck Millipore, Darmstadt, Germany) using the BioDot SF apparatus (Bio-Rad, Munich, Germany).

    Techniques: Western Blot, Synthesized, In Vitro, Mutagenesis, Purification, Recombinant

    Single and combined treatment with As 2 O 3  (As) and resveratrol (Res) altered the expression of apoptosis-related caspases. SK-N-SH cells were treated with 75 μg/ml resveratrol, 2 μM of As 2 O 3  or drugs combined for 48 h. Equal amounts of protein lysates from each sample were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) together with the protein markers, transferred onto polyvinylidene fluoride membranes, and subsequently blotted with specific anti-procaspase-3, anti-procaspase-9, anti-cleaved caspase-3, anticleaved caspase-9 and anti-β-actin antibodies for 2 h. The final signals for each specific protein on the same membranes were visualized by enhanced chemiluminescence kit and quantitated with the Image J software. Results represent the mean±S.D. of three independent experiments. *Significantly different at p

    Journal: Cancer Genomics & Proteomics

    Article Title: Novel Combination of Arsenic Trioxide (As2O3) Plus Resveratrol in Inducing Programmed Cell Death of Human Neuroblastoma SK-N-SH Cells

    doi: 10.21873/cgp.20104

    Figure Lengend Snippet: Single and combined treatment with As 2 O 3 (As) and resveratrol (Res) altered the expression of apoptosis-related caspases. SK-N-SH cells were treated with 75 μg/ml resveratrol, 2 μM of As 2 O 3 or drugs combined for 48 h. Equal amounts of protein lysates from each sample were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) together with the protein markers, transferred onto polyvinylidene fluoride membranes, and subsequently blotted with specific anti-procaspase-3, anti-procaspase-9, anti-cleaved caspase-3, anticleaved caspase-9 and anti-β-actin antibodies for 2 h. The final signals for each specific protein on the same membranes were visualized by enhanced chemiluminescence kit and quantitated with the Image J software. Results represent the mean±S.D. of three independent experiments. *Significantly different at p

    Article Snippet: The proteins of each sample were quantitated and equal amounts of proteins (40 μg) were separated on 12.5% acrylamide gels by sodium dodecyl sulfate (SDS) electrophoresis and then transferred to Immobilon-P Transfer Membrane (Merck Millipore, Billerica, MA, USA).

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

    Single and combined treatment with As 2 O 3  and resveratrol reduced the expression of B-cell lymphoma 2 (BCL2) family proteins. SK-N-SH cells were untreated, or treated with 75 μg/ml resveratrol alone, 2 μM As 2 O 3  alone or As 2 O 3  plus resveratrol for 48 h. Equal amounts of protein lysates from each sample were subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes, and subsequently immunoblotted with anti-BCL2, anti-BH3 interacting domain death agonist (BID), anti-B-cell lymphoma (BCL)-x/L, and anti-β-actin. The signals were visualized via enhanced chemiluminescence and quantitated with the Image J software. Results represent the mean±S.D. of three independent experiments. *Significantly different at p

    Journal: Cancer Genomics & Proteomics

    Article Title: Novel Combination of Arsenic Trioxide (As2O3) Plus Resveratrol in Inducing Programmed Cell Death of Human Neuroblastoma SK-N-SH Cells

    doi: 10.21873/cgp.20104

    Figure Lengend Snippet: Single and combined treatment with As 2 O 3 and resveratrol reduced the expression of B-cell lymphoma 2 (BCL2) family proteins. SK-N-SH cells were untreated, or treated with 75 μg/ml resveratrol alone, 2 μM As 2 O 3 alone or As 2 O 3 plus resveratrol for 48 h. Equal amounts of protein lysates from each sample were subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes, and subsequently immunoblotted with anti-BCL2, anti-BH3 interacting domain death agonist (BID), anti-B-cell lymphoma (BCL)-x/L, and anti-β-actin. The signals were visualized via enhanced chemiluminescence and quantitated with the Image J software. Results represent the mean±S.D. of three independent experiments. *Significantly different at p

    Article Snippet: The proteins of each sample were quantitated and equal amounts of proteins (40 μg) were separated on 12.5% acrylamide gels by sodium dodecyl sulfate (SDS) electrophoresis and then transferred to Immobilon-P Transfer Membrane (Merck Millipore, Billerica, MA, USA).

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

    Epitope identification through dot-blotting with synthetic peptides. (A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the PVDF membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using RG-M18 mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).

    Journal: PLoS ONE

    Article Title: Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection

    doi: 10.1371/journal.pone.0126121

    Figure Lengend Snippet: Epitope identification through dot-blotting with synthetic peptides. (A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the PVDF membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using RG-M18 mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).

    Article Snippet: After blocking with 5% skimmed milk in TBST buffer at 37°C for 30 min, the PVDF membrane was incubated with 1:1,000 diluted RG-M18 mAB in 5% skimmed milk at 37°C for 1 h. The membrane was then washed, and subjected to secondary antibody hybridization with polyclonal goat anti-mouse immunoglobulin/AP at a dilution of 1:5,000 in TBST buffer for 1 h at 37°C.

    Techniques: Peptide Microarray, Positive Control

    S100A9 is secreted under phosphorylated form. Validation of the custom-made phospho-specific S100A9 antibody (A,B) . (A) Non-stimulated cell lysates (CTL), fMLF-stimulated (100 nM, 10 min), phosphatase (CiP)-treated fMLF-stimulated cell lysates as well as recombinant S100A8 and S100A9 (2 μg) were run on a Tris-Tricine gel (10% acrylamide), transferred to PVDF membrane and probed with the anti-PhosphoS100A9 antibody and with the Mac387 antibody to detect total S100A8 and S100A9. (B) 3 μg of purified S100A8/A9 (lane 1) and S100A8/PhosphoA9 (lane 2) were run on a Tris-Tricine gel (10% acrylamide), transferred to PVDF membrane and probed with anti-PhosphoS100A9 antibody alone (a), preincubated 1 h with 1 μg/mL (b) or 4 μg/mL (c) of immunogenic peptide. The same membrane was also probed with B-5 and EPR3554 antibody in order to detect total S100A8 and S100A9 (d–f). (C) Supernatants (SNs) of control or PMA-stimulated dHL60 cells or purified neutrophils were concentrated with Centricon ® devices (cut-off 3 kDa) and then loaded on a 10% Tris-Tricine Gel. Western blots were probed with the anti-PhosphoS100A9 antibody and total S100A9 was detected by the Mac387 antibody.

    Journal: Frontiers in Immunology

    Article Title: Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9

    doi: 10.3389/fimmu.2018.00447

    Figure Lengend Snippet: S100A9 is secreted under phosphorylated form. Validation of the custom-made phospho-specific S100A9 antibody (A,B) . (A) Non-stimulated cell lysates (CTL), fMLF-stimulated (100 nM, 10 min), phosphatase (CiP)-treated fMLF-stimulated cell lysates as well as recombinant S100A8 and S100A9 (2 μg) were run on a Tris-Tricine gel (10% acrylamide), transferred to PVDF membrane and probed with the anti-PhosphoS100A9 antibody and with the Mac387 antibody to detect total S100A8 and S100A9. (B) 3 μg of purified S100A8/A9 (lane 1) and S100A8/PhosphoA9 (lane 2) were run on a Tris-Tricine gel (10% acrylamide), transferred to PVDF membrane and probed with anti-PhosphoS100A9 antibody alone (a), preincubated 1 h with 1 μg/mL (b) or 4 μg/mL (c) of immunogenic peptide. The same membrane was also probed with B-5 and EPR3554 antibody in order to detect total S100A8 and S100A9 (d–f). (C) Supernatants (SNs) of control or PMA-stimulated dHL60 cells or purified neutrophils were concentrated with Centricon ® devices (cut-off 3 kDa) and then loaded on a 10% Tris-Tricine Gel. Western blots were probed with the anti-PhosphoS100A9 antibody and total S100A9 was detected by the Mac387 antibody.

    Article Snippet: Samples were run on a Tris-Tricine gel (10% acrylamide) and the proteins were then electrotransferred to a PVDF membrane (Merck-Millipore, Overijse, Belgium).

    Techniques: CTL Assay, Recombinant, Purification, Western Blot