polyvinylidene difluoride pvdf membranes  (Thermo Fisher)


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    PVDF Transfer Membrane x
    Description:
    Thermo Scientific Pierce PVDF Transfer Membranes are made of high quality polyvinylidene difluoride and provide high binding capacity for proteins and nucleic acids for Western Southern and Northern blotting methods This PVDF membrane has a 0 45 micron pore size and is ideal for colorimetric and chemiluminescent detection Features of PVDF membranes • Western blotting PVDF transfer membranes manufactured especially for protein transfer and Western blot applications • Compatible and durable polyvinyl difluoride PVDF is compatible with most organic solvents acids and mild bases doesn t tear or become brittle like nitrocellulose • Specialized use the 0 45µm variety for colorimetric and chemiluminescent detection Our PVDF polyvinyl difluoride membranes are more resistant to discoloration than other commercially available PVDF membranes and have better retention of adsorbed proteins than other types of transfer membranes used for Western blotting including nitrocellulose This membrane has lower background levels and increased sensitivity for fluorescent probing than regular PVDF or nitrocellulose membranes Related Products PVDF Transfer Membrane 0 45 µm 10 cm x 10 cm Low Fluorescence PVDF Transfer Membrane 0 2 µm 7 cm x 8 4 cm PVDF Transfer Membrane 0 2 µm 26 5 cm x 3 75 m
    Catalog Number:
    88518
    Price:
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    Applications:
    Protein Assays and Analysis|Protein Biology|Western Blotting
    Category:
    Gels Fractionation Strips Membranes
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    Structured Review

    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.
    Thermo Scientific Pierce PVDF Transfer Membranes are made of high quality polyvinylidene difluoride and provide high binding capacity for proteins and nucleic acids for Western Southern and Northern blotting methods This PVDF membrane has a 0 45 micron pore size and is ideal for colorimetric and chemiluminescent detection Features of PVDF membranes • Western blotting PVDF transfer membranes manufactured especially for protein transfer and Western blot applications • Compatible and durable polyvinyl difluoride PVDF is compatible with most organic solvents acids and mild bases doesn t tear or become brittle like nitrocellulose • Specialized use the 0 45µm variety for colorimetric and chemiluminescent detection Our PVDF polyvinyl difluoride membranes are more resistant to discoloration than other commercially available PVDF membranes and have better retention of adsorbed proteins than other types of transfer membranes used for Western blotting including nitrocellulose This membrane has lower background levels and increased sensitivity for fluorescent probing than regular PVDF or nitrocellulose membranes Related Products PVDF Transfer Membrane 0 45 µm 10 cm x 10 cm Low Fluorescence PVDF Transfer Membrane 0 2 µm 7 cm x 8 4 cm PVDF Transfer Membrane 0 2 µm 26 5 cm x 3 75 m
    https://www.bioz.com/result/polyvinylidene difluoride pvdf membranes/product/Thermo Fisher
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    polyvinylidene difluoride pvdf membranes - by Bioz Stars, 2020-12
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    Images

    1) Product Images from "Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004347

    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.
    Figure Legend 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.

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

    2) Product Images from "Targeting Swine Leukocyte Antigen Class I Molecules for Proteasomal Degradation by the nsp1α Replicase Protein of the Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06"

    Article Title: Targeting Swine Leukocyte Antigen Class I Molecules for Proteasomal Degradation by the nsp1α Replicase Protein of the Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06

    Journal: Journal of Virology

    doi: 10.1128/JVI.02307-15

    HP-PRRSV promotes SLA-I degradation via the ubiquitin-proteasome pathway. (A) PAMs were either mock infected or infected with HP-PRRSV at an MOI of 1. At 4 h postinfection, the cells were treated with MG132 or DMSO for 8 h and then processed and subjected to Western blot analysis. (B) PRRSV- or mock-infected PAMs were treated with 5 μM MG132 for 8 h at 4 h postinfection. The cells were then lysed and subjected to immunoprecipitation (IP) with rabbit polyclonal antibodies to SLA-I-HC or anti-β2m in conjunction with protein A-Sepharose beads, respectively. The proteins bound to the beads were separated by SDS-PAGE, transferred onto a PVDF membrane, and subjected to immunoblotting (IB) with antibodies to SLA-I-HC, β2m, or ubiquitin (Ub). (C) Mock- or PRRSV-infected PAMs were treated with Z-VAD-FMK (200 μM) or DMSO for 12 h. At 12 h postinfection, the cells were harvested and subjected to SDS-PAGE and Western blot analysis with the proper antibodies.
    Figure Legend Snippet: HP-PRRSV promotes SLA-I degradation via the ubiquitin-proteasome pathway. (A) PAMs were either mock infected or infected with HP-PRRSV at an MOI of 1. At 4 h postinfection, the cells were treated with MG132 or DMSO for 8 h and then processed and subjected to Western blot analysis. (B) PRRSV- or mock-infected PAMs were treated with 5 μM MG132 for 8 h at 4 h postinfection. The cells were then lysed and subjected to immunoprecipitation (IP) with rabbit polyclonal antibodies to SLA-I-HC or anti-β2m in conjunction with protein A-Sepharose beads, respectively. The proteins bound to the beads were separated by SDS-PAGE, transferred onto a PVDF membrane, and subjected to immunoblotting (IB) with antibodies to SLA-I-HC, β2m, or ubiquitin (Ub). (C) Mock- or PRRSV-infected PAMs were treated with Z-VAD-FMK (200 μM) or DMSO for 12 h. At 12 h postinfection, the cells were harvested and subjected to SDS-PAGE and Western blot analysis with the proper antibodies.

    Techniques Used: Infection, Western Blot, Immunoprecipitation, SDS Page

    nsp1α interacts with both chains of SLA-I and promotes their degradation through the ubiquitin-proteasome pathway. (A) Summary of the nsp1α constructs used in this study. (B to D) HEK 293T cells transfected with the plasmid expressing FLAG-SLA-I-HC (B) or Myc-β2m (C and D) together with the plasmid expressing either HA-nsp1α or its derivatives. At 24 h posttransfection, the cells were treated with 5 μM MG132 or DMSO for 12 h. At 36 h posttransfection, the cells were either subjected to direct Western blot analyses (B, left, C, and D, left) or lysed and immunoprecipitated with anti-FLAG (B) or Myc (D) antibodies. The proteins bound to Sepharose beads were separated by SDS-PAGE, transferred onto a PVDF membrane, and probed with the antibodies to FLAG, HA, or ubiquitin (right). β-Actin served as a loading control. The data are representative of results from three independent experiments.
    Figure Legend Snippet: nsp1α interacts with both chains of SLA-I and promotes their degradation through the ubiquitin-proteasome pathway. (A) Summary of the nsp1α constructs used in this study. (B to D) HEK 293T cells transfected with the plasmid expressing FLAG-SLA-I-HC (B) or Myc-β2m (C and D) together with the plasmid expressing either HA-nsp1α or its derivatives. At 24 h posttransfection, the cells were treated with 5 μM MG132 or DMSO for 12 h. At 36 h posttransfection, the cells were either subjected to direct Western blot analyses (B, left, C, and D, left) or lysed and immunoprecipitated with anti-FLAG (B) or Myc (D) antibodies. The proteins bound to Sepharose beads were separated by SDS-PAGE, transferred onto a PVDF membrane, and probed with the antibodies to FLAG, HA, or ubiquitin (right). β-Actin served as a loading control. The data are representative of results from three independent experiments.

    Techniques Used: Construct, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, SDS Page

    3) Product Images from "A Mouse Strain Defective in Both T Cells and NK Cells Has Enhanced Sensitivity to Tumor Induction by Plasmid DNA Expressing Both Activated H-Ras and c-Myc"

    Article Title: A Mouse Strain Defective in Both T Cells and NK Cells Has Enhanced Sensitivity to Tumor Induction by Plasmid DNA Expressing Both Activated H-Ras and c-Myc

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108926

    All tumor cell lines expressed both the H-Ras and the c-Myc proteins. Lysates of the tumor cell lines corresponding to 2.5 µg of protein for H-Ras detection and 30 µg of protein for c-Myc detection were resolved by SDS-PAGE on 4–20% gradient gels and the proteins revealed by western analysis. A B : c-Myc. C D : H-Ras. A C : Lines from tumors induced by Sca I linear plasmid; B D : Lines from tumors induced by Eco RI linear plasmid. Lanes a: NIH/3T3 mouse control cell line. Lanes b to n represent different tumor-cell lines; these lines correspond to the cell lines in Fig. 6 and Fig. 7 . Membranes were subsequently reacted with an antibody to actin to assess protein loadings. The PVDF membranes were reacted with the anti-actin antibody sc-1616; a band migrating at approximately 43 kD is seen in all lanes.
    Figure Legend Snippet: All tumor cell lines expressed both the H-Ras and the c-Myc proteins. Lysates of the tumor cell lines corresponding to 2.5 µg of protein for H-Ras detection and 30 µg of protein for c-Myc detection were resolved by SDS-PAGE on 4–20% gradient gels and the proteins revealed by western analysis. A B : c-Myc. C D : H-Ras. A C : Lines from tumors induced by Sca I linear plasmid; B D : Lines from tumors induced by Eco RI linear plasmid. Lanes a: NIH/3T3 mouse control cell line. Lanes b to n represent different tumor-cell lines; these lines correspond to the cell lines in Fig. 6 and Fig. 7 . Membranes were subsequently reacted with an antibody to actin to assess protein loadings. The PVDF membranes were reacted with the anti-actin antibody sc-1616; a band migrating at approximately 43 kD is seen in all lanes.

    Techniques Used: SDS Page, Western Blot, Plasmid Preparation

    4) Product Images from "Murine splenic B cells express corticotropin-releasing hormone receptor 2 that affect their viability during a stress response"

    Article Title: Murine splenic B cells express corticotropin-releasing hormone receptor 2 that affect their viability during a stress response

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18401-y

    Murine splenic B cells express CRHR2 receptors. Splenocytes were incubated or not with CRH 1 or 100 nM for 48 h. Then, B cells were isolated by negative selection. ( a ) Total RNA extracted from hippocampus (positive control) and murine splenic B cells was reverse transcribed. Then, PCR amplifications were carried out using CRHR1 or CRHR2 specific primer pairs. Lanes 1 and 4: CRHR1 and CRHR2 amplification products obtained from hippocampus total RNA (positive controls). Lanes 2 and 5: amplification products obtained from splenic B cell total RNA. Lanes 3 and 6: negative controls (no cDNA). Full-length gel is shown in Supplementary Fig. S1 . ( b ) Western blotting analyses. B cell protein lysates were loaded on a SDS-page gel, submitted to electrophoresis and transferred onto a PVDF membrane. The membrane was incubated with an anti-CRHR antibody, stripped and rehybridized with an anti-GAPDH antibody. Panels show bands revealed by both antibodies on the same membrane. Full-length blots are shown in Supplementary Fig. S1 . ( c ) Immunofluorescence analysis of isolated splenic B cells labeled with anti-CD19 (red) and anti-CRHR (green) antibodies. Panels (a), (c) and (d) are representative of three independent experiments.
    Figure Legend Snippet: Murine splenic B cells express CRHR2 receptors. Splenocytes were incubated or not with CRH 1 or 100 nM for 48 h. Then, B cells were isolated by negative selection. ( a ) Total RNA extracted from hippocampus (positive control) and murine splenic B cells was reverse transcribed. Then, PCR amplifications were carried out using CRHR1 or CRHR2 specific primer pairs. Lanes 1 and 4: CRHR1 and CRHR2 amplification products obtained from hippocampus total RNA (positive controls). Lanes 2 and 5: amplification products obtained from splenic B cell total RNA. Lanes 3 and 6: negative controls (no cDNA). Full-length gel is shown in Supplementary Fig. S1 . ( b ) Western blotting analyses. B cell protein lysates were loaded on a SDS-page gel, submitted to electrophoresis and transferred onto a PVDF membrane. The membrane was incubated with an anti-CRHR antibody, stripped and rehybridized with an anti-GAPDH antibody. Panels show bands revealed by both antibodies on the same membrane. Full-length blots are shown in Supplementary Fig. S1 . ( c ) Immunofluorescence analysis of isolated splenic B cells labeled with anti-CD19 (red) and anti-CRHR (green) antibodies. Panels (a), (c) and (d) are representative of three independent experiments.

    Techniques Used: Incubation, Isolation, Selection, Positive Control, Polymerase Chain Reaction, Amplification, Western Blot, SDS Page, Electrophoresis, Immunofluorescence, Labeling

    5) Product Images from "Microglia Are Critical in Host Defense against Prion Disease"

    Article Title: Microglia Are Critical in Host Defense against Prion Disease

    Journal: Journal of Virology

    doi: 10.1128/JVI.00549-18

    Western blot and densitometry of PrPres in the brains of PLX5622-treated and untreated mice infected with scrapie. Mice infected with scrapie strain RML were treated with PLX5622 or untreated and euthanized at 80 dpi (A), at 100 dpi (B), or at clinical endpoint (terminal) (C). Brain homogenates were digested with proteinase K, and proteins were separated by SDS-PAGE and transferred to PVDF membranes. Blocked membranes were probed with anti-PrP (D13), and bands were visualized by chemiluminescence. (A and B) Approximately 5-s exposures to film using SuperSignal West Femto substrate; (C) 10-min exposure to film using ECL Western blotting substrate. The days postinfection of euthanasia of each mouse is given below the immunoblots in panel C. Densitometry on each lane was performed to assess differences between the groups, and plots of the adjusted volumes are shown to the right of the immunoblots. Statistical analysis was performed using a two-tailed t test comparing PLX5622-treated to untreated animals. P values are indicated.
    Figure Legend Snippet: Western blot and densitometry of PrPres in the brains of PLX5622-treated and untreated mice infected with scrapie. Mice infected with scrapie strain RML were treated with PLX5622 or untreated and euthanized at 80 dpi (A), at 100 dpi (B), or at clinical endpoint (terminal) (C). Brain homogenates were digested with proteinase K, and proteins were separated by SDS-PAGE and transferred to PVDF membranes. Blocked membranes were probed with anti-PrP (D13), and bands were visualized by chemiluminescence. (A and B) Approximately 5-s exposures to film using SuperSignal West Femto substrate; (C) 10-min exposure to film using ECL Western blotting substrate. The days postinfection of euthanasia of each mouse is given below the immunoblots in panel C. Densitometry on each lane was performed to assess differences between the groups, and plots of the adjusted volumes are shown to the right of the immunoblots. Statistical analysis was performed using a two-tailed t test comparing PLX5622-treated to untreated animals. P values are indicated.

    Techniques Used: Western Blot, Mouse Assay, Infection, SDS Page, Two Tailed Test

    6) Product Images from "Drebrin contains a cryptic F-actin-bundling activity regulated by Cdk5 phosphorylation"

    Article Title: Drebrin contains a cryptic F-actin-bundling activity regulated by Cdk5 phosphorylation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201303005

    In vitro F-actin cosedimentation assays show that drebrin is an F-actin–bundling protein and confirm the presence of separate F-actin binding in the CC and Hel domains. Actin filaments were incubated with or without recombinant drebrin proteins and F-actin bundles/meshes pelleted by low speed centrifugation (P L ). The resulting supernatant was then centrifuged at high speed to pellet single actin filaments (P H ). The supernatants from the high-speed centrifugation (S), P L , and P H were analyzed by gel electrophoresis and Coomassie blue staining. (A) Drebrin bundles and binds to F-actin. Actin filaments pellet after high-speed centrifugation (P H ) but do not pellet after low-speed centrifugation (P L ). Addition of drebrin results in the appearance of actin in the low-speed pellet (P L ) together with a small amount of drebrin as well as in the high-speed pellet (P H ). Drebrin alone does not pellet under either condition. (B) The CC domain binds but does not bundle F-actin. When added to F-actin, the CC construct appears in the high-speed pellet (P H ) but not the low-speed pellet (P L ). The CC construct alone does not pellet under either condition. (C) The CC-Hel domain construct is a strong F-actin bundler. When added to F-actin, the CC-Hel construct appears in both the high-speed pellet (P H ) and the low-speed pellet (P L ). The CC-Hel construct alone does not pellet under either condition. (D) Addition of the PP domain has little effect on the bundling activity of the CC-Hel domains. When added to F-actin, the CC-Hel-PP construct appears in both the high-speed pellet (P H ) and the low-speed pellet (P L ). The CC-Hel-PP construct alone does not pellet under either condition. (E–H) Electron micrographs of negatively stained F-actin from in vitro F-actin cosedimentation assays after addition of drebrin or drebrin deletion constructs. (E) After the addition of drebrin, thin, loose bundles of F-actin are visible. Bar, 50 nm. (F) After the addition of the CC-Hel construct, thick, tight bundles of F-actin are visible. Bar, 20 nm. (G) After the addition of fascin, a thick, tight bundle of F-actin is visible. Bar, 20 nm. (H) After the addition of the CC construct, single actin filaments are seen and there are no bundles of F-actin. Bar, 200 nm. (I) The BB domain binds to the ADFH domain. An immunoblot of YFP-tagged proteins, identified with GFP antibody, in BB-His pull-downs (Pull-down) is shown. Cell extracts of COS-7 cells expressing drebrin deletion constructs (Input) were mixed with recombinant BB-His and proteins bound to nickel beads separated by SDS-PAGE and transferred to PVDF membranes. Blots were probed with GFP and His-tag antibodies. (J) Electron micrograph of negatively stained F-actin from an in vitro F-actin cosedimentation assay after addition of drebrin that had been preincubated with a neonatal rat brain extract. A thin, tight bundle of F-actin is visible. Bar, 50 nm. (K) The BB domain inhibits binding of the ADFH-CC construct to F-actin. Actin filaments were incubated with the His-tagged ADFH-CC construct in the absence or presence of increasing concentrations ([BB-His] µM) of the His-tagged BB domain. The ratio of His-tagged ADFH-CC to actin is shown (ADFH-CC/actin). (L) The BB domain does not inhibit binding of the S142D ADFH-CC construct to F-actin. Actin filaments were incubated with the His-tagged S142D ADFH-CC construct in the absence or presence of increasing concentrations ([BB-His] µM) of the His-tagged BB domain. The ratio of His-tagged ADFH-CC to actin is shown (ADFH-CC/actin).
    Figure Legend Snippet: In vitro F-actin cosedimentation assays show that drebrin is an F-actin–bundling protein and confirm the presence of separate F-actin binding in the CC and Hel domains. Actin filaments were incubated with or without recombinant drebrin proteins and F-actin bundles/meshes pelleted by low speed centrifugation (P L ). The resulting supernatant was then centrifuged at high speed to pellet single actin filaments (P H ). The supernatants from the high-speed centrifugation (S), P L , and P H were analyzed by gel electrophoresis and Coomassie blue staining. (A) Drebrin bundles and binds to F-actin. Actin filaments pellet after high-speed centrifugation (P H ) but do not pellet after low-speed centrifugation (P L ). Addition of drebrin results in the appearance of actin in the low-speed pellet (P L ) together with a small amount of drebrin as well as in the high-speed pellet (P H ). Drebrin alone does not pellet under either condition. (B) The CC domain binds but does not bundle F-actin. When added to F-actin, the CC construct appears in the high-speed pellet (P H ) but not the low-speed pellet (P L ). The CC construct alone does not pellet under either condition. (C) The CC-Hel domain construct is a strong F-actin bundler. When added to F-actin, the CC-Hel construct appears in both the high-speed pellet (P H ) and the low-speed pellet (P L ). The CC-Hel construct alone does not pellet under either condition. (D) Addition of the PP domain has little effect on the bundling activity of the CC-Hel domains. When added to F-actin, the CC-Hel-PP construct appears in both the high-speed pellet (P H ) and the low-speed pellet (P L ). The CC-Hel-PP construct alone does not pellet under either condition. (E–H) Electron micrographs of negatively stained F-actin from in vitro F-actin cosedimentation assays after addition of drebrin or drebrin deletion constructs. (E) After the addition of drebrin, thin, loose bundles of F-actin are visible. Bar, 50 nm. (F) After the addition of the CC-Hel construct, thick, tight bundles of F-actin are visible. Bar, 20 nm. (G) After the addition of fascin, a thick, tight bundle of F-actin is visible. Bar, 20 nm. (H) After the addition of the CC construct, single actin filaments are seen and there are no bundles of F-actin. Bar, 200 nm. (I) The BB domain binds to the ADFH domain. An immunoblot of YFP-tagged proteins, identified with GFP antibody, in BB-His pull-downs (Pull-down) is shown. Cell extracts of COS-7 cells expressing drebrin deletion constructs (Input) were mixed with recombinant BB-His and proteins bound to nickel beads separated by SDS-PAGE and transferred to PVDF membranes. Blots were probed with GFP and His-tag antibodies. (J) Electron micrograph of negatively stained F-actin from an in vitro F-actin cosedimentation assay after addition of drebrin that had been preincubated with a neonatal rat brain extract. A thin, tight bundle of F-actin is visible. Bar, 50 nm. (K) The BB domain inhibits binding of the ADFH-CC construct to F-actin. Actin filaments were incubated with the His-tagged ADFH-CC construct in the absence or presence of increasing concentrations ([BB-His] µM) of the His-tagged BB domain. The ratio of His-tagged ADFH-CC to actin is shown (ADFH-CC/actin). (L) The BB domain does not inhibit binding of the S142D ADFH-CC construct to F-actin. Actin filaments were incubated with the His-tagged S142D ADFH-CC construct in the absence or presence of increasing concentrations ([BB-His] µM) of the His-tagged BB domain. The ratio of His-tagged ADFH-CC to actin is shown (ADFH-CC/actin).

    Techniques Used: In Vitro, Binding Assay, Incubation, Recombinant, Centrifugation, Nucleic Acid Electrophoresis, Staining, Construct, Activity Assay, Expressing, SDS Page

    7) Product Images from "A Cell Culture System for the Study of Amyloid Pathogenesis "

    Article Title: A Cell Culture System for the Study of Amyloid Pathogenesis

    Journal: The American Journal of Pathology

    doi:

    SDS-PAGE of cell layers harvested from amyloid-laden cultures showing C-terminal cleavage of SAA. Cultures were rinsed with RPMI, scraped into PBS, pelleted by centrifugation, and solubilized in SDS gel-loading buffer. A: Coomassie blue-stained PVDF membrane. Lane 1: human AA protein isolated from spleen. Lane 2: Cell layer with amyloid (14-day culture). Lane 3: purified rSAA2. B: Immunoblot of A , probed with anti-mouse SAA antiserum. Lane 1: cell layer containing SAA and AA peptides (dimers of SAA and AA are also present). Lane 2: Purified rSAA2. C: Coomassie blue-stained PVDF membrane. Lane 1: Human AA protein from spleen. Lane 2: Cell layer with amyloid (19-day culture). Thin lines at right indicate peptides subjected to N-terminal sequencing.
    Figure Legend Snippet: SDS-PAGE of cell layers harvested from amyloid-laden cultures showing C-terminal cleavage of SAA. Cultures were rinsed with RPMI, scraped into PBS, pelleted by centrifugation, and solubilized in SDS gel-loading buffer. A: Coomassie blue-stained PVDF membrane. Lane 1: human AA protein isolated from spleen. Lane 2: Cell layer with amyloid (14-day culture). Lane 3: purified rSAA2. B: Immunoblot of A , probed with anti-mouse SAA antiserum. Lane 1: cell layer containing SAA and AA peptides (dimers of SAA and AA are also present). Lane 2: Purified rSAA2. C: Coomassie blue-stained PVDF membrane. Lane 1: Human AA protein from spleen. Lane 2: Cell layer with amyloid (19-day culture). Thin lines at right indicate peptides subjected to N-terminal sequencing.

    Techniques Used: SDS Page, Centrifugation, SDS-Gel, Staining, Isolation, Purification, Sequencing

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    Article Snippet: .. The hyper- and hypo-phosphorylated Cp were resolved by electrophoresis in a NuPAGE 12% Bis-Tris Protein Gel (Invitrogen) and transferred onto PVDF membrane (Invitrogen). ..

    Western Blot:

    Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations
    Article Snippet: .. SDS-PAGE with 11%-resolving/5%-stacking acrylamide gels, and WB on PVDF-membrane (Cat.#88518; ThermoFisher Scientific) were performed according to the standard protocol of the equipment-manufacturer (Bio-Rad Laboratories). .. Each PVDF-membrane piece (per antibody) was separately imaged via the auto-exposure function of the ChemiDoc-MP imaging system (Bio-Rad Laboratories).

    Article Title: Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR
    Article Snippet: .. The gel was transferred to a PVDF membrane by iBLOT (Invitrogen, Burlington, Ontario, Canada) and protein was detected by Western blot analysis (one-hour Western kit with TMB Substrate, GenScript) using Pierce 6x His epitope-tag IgG2b mouse antibody (ThermoFisher, Unionville, Ontario, Canada) . .. The Western blot was imaged using UVP ChemiDoc-It TS2, and the bands were analyzed using the software GelAnalyzer 19.1 (GelAnalyzer, Budapest, Hungary).

    Sequencing:

    Article Title: Factor VII activating protease (FSAP) promotes the proteolysis and inhibition of tissue factor pathway inhibitor (TFPI)
    Article Snippet: .. In other experiments cleaved TFPI was transferred to PVDF membranes and subjected to N-terminal sequencing (Applied Biosystems, Darmstadt, Germany) for the determination of the cleavage sites. .. After treatment of rTFPI (12.5 nM) with WT and MI FSAP, residual TFPI activity was measured with a TFPI chromogenic activity assay (Actichrome, American Diagnostica).

    SDS Page:

    Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations
    Article Snippet: .. SDS-PAGE with 11%-resolving/5%-stacking acrylamide gels, and WB on PVDF-membrane (Cat.#88518; ThermoFisher Scientific) were performed according to the standard protocol of the equipment-manufacturer (Bio-Rad Laboratories). .. Each PVDF-membrane piece (per antibody) was separately imaged via the auto-exposure function of the ChemiDoc-MP imaging system (Bio-Rad Laboratories).

    Article Title: Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability
    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). .. Proteins were revealed using the following antibodies at the indicated dilutions: i) polyclonal anti-MPK10 antibody, generated by rabbit immunization using recombinant MPK10 protein produced in E. coli transformed with pGEX-Strep3-MPK10 plasmid (Eurogentec), 1∶10,000; ii) anti-phospho-tyrosine antibody 4G10 Platinum from Millipore, 1∶1,000; and iii) secondary goat anti-rabbit-HRP and anti-mouse-HRP antibodies from Thermo Scientific, 1∶20,000.

    Article Title: Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs
    Article Snippet: .. Whole cell-lysates were separated by SDS-PAGE, electroblotted onto PVDF membranes, and reacted with O139-antigen–specific antibodies (Remel, http://www.remelinc.com ). .. For signal detection, Protein A-HRP conjugate (Bio-Rad, http://www.bio-rad.com ) and Lumi-Light Western Blotting substrate (Roche Applied Science, http://www.roche-applied-science.com ) were used.

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    Thermo Fisher pvdf membrane
    Inhibition of RAS stops wt-RAS sequence Philadelphia-like ALL cell growth in the presence of TSLP. (A) MUTZ-5 cells were seeded at 6.5×10 5 /mL density and cultured over 4 days with either 0.5% DMSO (vehicle control), 50 µM Salirasib (indirect Pan-RAS inh.), 10 µM PI-103 (PI3K/mTOR dual inh.), or 5 µM Ruxolitinib (JAK inh.), each in absence or presence of 20 ng/mL human TSLP. Cell count and viability (percentage of acridine orange-positive cells not stained by 4’,6-diamidino-2-phenylindole (DAPI) was determined in a NC-250 automated cell counter daily. The stacked-bar graph on the left side shows the growth rate after the 90 hrs timepoint, averaged from 2 independent experiments, each with triplicate wells. Red error bars are SD from the dead cell fraction while the black error bars show the SD of the viable cells. P -values were calculated in one-way ANOVA from the total cell growth rate and adjusted in a post-hoc Bonferroni multiple comparison. Only relevant P -values are shown in the graph, for a complete list see Supplementary-Tab.S2. (B) The graph shows the cell viability of the experiment in (A) over time. (C) MUTZ-5 cells were pre-treated for 2 hrs with either 0.5% DMSO (vehicle control), 10 µM PI-103 (PI3K/mTOR dual inh.), 50 µM Salirasib (indirect Pan-RAS inh.), 5 µM Ruxolitinib (JAK inh.), 50 µM Vemurafenib (Pan-Raf inh.), or 25 µM II-B08 (PTPN11 inh.), and then stimulated with 20 ng/mL human TSLP for 10 min followed by cell lysis. Each lysate sample was split up for analysis in RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down (left) and lysate samples (right) were loaded on separate gels. An <t>SDS-PAGE</t> followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same <t>PVDF-membrane,</t> membranes were stripped and reprobed with new antibodies. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band.
    Pvdf Membrane, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Top Panel: <t>SDS-PAGE</t> analysis of time course digestion of Vip3Ab1 and Vip3Bc1 with H. zea and P. includens gut enzymes. Vip3Ab1 and Vip3Bc1 proteins (150 µg/mL) were incubated with gut fluids from H. zea (left) and P. includens (right) at 30 °C for various time intervals at pH 10.0. Bottom Panel: SDS-PAGE analysis of overnight digestion of Vip3 chimeras with H. zea gut enzymes. Vip3_AB and Vip3_BA proteins (110 µg/ml) were incubated with H. zea gut fluids for 16 hours at 30 °C in a total volume of 100 µL at pH 10.0. All reactions were stopped with protease inhibitors and 30 µL of the reaction loaded as described in Materials and Methods. Equivalent lanes were loaded and blotted onto a <t>PVDF</t> membrane for N-terminal sequencing.
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    TNF-α induces formation of <t>SDS-stable</t> complexes between APP-βCTF and HS-anMan in growing mouse N2a neuroblastoma cells. The cells were either untreated (−) or grown for 48 h in the presence of 100 pg/ml of TNF-α (+). Immunoprecipitation (IP) was performed on the cell extracts with pAb A8717, which recognizes the C-terminus of APP. The same amount of protein was applied to each lane, and electrophoresis was followed by transfer to <t>PVDF</t> membranes and western blotting using either mAb 4G8 (for Aβ) or mAb AM (for HS-anMan). In lane 1, the primary antibody was omitted (0). A band corresponding to APP as in lane 4 was also seen when IP was performed with pAb Aβ40 (not shown).
    Pierce Pvdf Membranes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Inhibition of RAS stops wt-RAS sequence Philadelphia-like ALL cell growth in the presence of TSLP. (A) MUTZ-5 cells were seeded at 6.5×10 5 /mL density and cultured over 4 days with either 0.5% DMSO (vehicle control), 50 µM Salirasib (indirect Pan-RAS inh.), 10 µM PI-103 (PI3K/mTOR dual inh.), or 5 µM Ruxolitinib (JAK inh.), each in absence or presence of 20 ng/mL human TSLP. Cell count and viability (percentage of acridine orange-positive cells not stained by 4’,6-diamidino-2-phenylindole (DAPI) was determined in a NC-250 automated cell counter daily. The stacked-bar graph on the left side shows the growth rate after the 90 hrs timepoint, averaged from 2 independent experiments, each with triplicate wells. Red error bars are SD from the dead cell fraction while the black error bars show the SD of the viable cells. P -values were calculated in one-way ANOVA from the total cell growth rate and adjusted in a post-hoc Bonferroni multiple comparison. Only relevant P -values are shown in the graph, for a complete list see Supplementary-Tab.S2. (B) The graph shows the cell viability of the experiment in (A) over time. (C) MUTZ-5 cells were pre-treated for 2 hrs with either 0.5% DMSO (vehicle control), 10 µM PI-103 (PI3K/mTOR dual inh.), 50 µM Salirasib (indirect Pan-RAS inh.), 5 µM Ruxolitinib (JAK inh.), 50 µM Vemurafenib (Pan-Raf inh.), or 25 µM II-B08 (PTPN11 inh.), and then stimulated with 20 ng/mL human TSLP for 10 min followed by cell lysis. Each lysate sample was split up for analysis in RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down (left) and lysate samples (right) were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, membranes were stripped and reprobed with new antibodies. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band.

    Journal: bioRxiv

    Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

    doi: 10.1101/2020.02.03.931725

    Figure Lengend Snippet: Inhibition of RAS stops wt-RAS sequence Philadelphia-like ALL cell growth in the presence of TSLP. (A) MUTZ-5 cells were seeded at 6.5×10 5 /mL density and cultured over 4 days with either 0.5% DMSO (vehicle control), 50 µM Salirasib (indirect Pan-RAS inh.), 10 µM PI-103 (PI3K/mTOR dual inh.), or 5 µM Ruxolitinib (JAK inh.), each in absence or presence of 20 ng/mL human TSLP. Cell count and viability (percentage of acridine orange-positive cells not stained by 4’,6-diamidino-2-phenylindole (DAPI) was determined in a NC-250 automated cell counter daily. The stacked-bar graph on the left side shows the growth rate after the 90 hrs timepoint, averaged from 2 independent experiments, each with triplicate wells. Red error bars are SD from the dead cell fraction while the black error bars show the SD of the viable cells. P -values were calculated in one-way ANOVA from the total cell growth rate and adjusted in a post-hoc Bonferroni multiple comparison. Only relevant P -values are shown in the graph, for a complete list see Supplementary-Tab.S2. (B) The graph shows the cell viability of the experiment in (A) over time. (C) MUTZ-5 cells were pre-treated for 2 hrs with either 0.5% DMSO (vehicle control), 10 µM PI-103 (PI3K/mTOR dual inh.), 50 µM Salirasib (indirect Pan-RAS inh.), 5 µM Ruxolitinib (JAK inh.), 50 µM Vemurafenib (Pan-Raf inh.), or 25 µM II-B08 (PTPN11 inh.), and then stimulated with 20 ng/mL human TSLP for 10 min followed by cell lysis. Each lysate sample was split up for analysis in RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down (left) and lysate samples (right) were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, membranes were stripped and reprobed with new antibodies. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band.

    Article Snippet: SDS-PAGE with 11%-resolving/5%-stacking acrylamide gels, and WB on PVDF-membrane (Cat.#88518; ThermoFisher Scientific) were performed according to the standard protocol of the equipment-manufacturer (Bio-Rad Laboratories).

    Techniques: Inhibition, Sequencing, Cell Culture, Cell Counting, Staining, Lysis, Pull Down Assay, SDS Page, Western Blot, Labeling

    Human Ph-like B-ALL (spontaneous CRLF2-rearrangment and JAK2R683G mutation) cells activate wildtype RAS and RAS-interacting proteins upon TSLP induction. MUTZ-5 cells (Human Ph-like B-ALL cells bearing CRLF2-rearranged and spontaneous JAK2R683G mutation) were stimulated with 20 ng/mL human TSLP (maximal effective TSLP-concentration, Supplementary-Fig.S3B) for 10 min before cell lysis. Each cell lysate was split up for analysis in RAS-GTP pull-down assay and for total protein signal. (A) RAS-GTP pull-down and lysate samples were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, each membrane part was stripped and reprobed with new antibodies. RAS-GTP pull-down samples are on the left side while the right-hand side blots show whole cell lysates of the same samples. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band. The grey arrow shows the unspecific signal of the GST-RAS binding domain (RBD) used in the active RAS pull-down assay acting as a loading control. The experiment was repeated 5 times independently and the graphs show the quantification for active RAS (RAS-GTP), phosphorylated MEK1/2 (phospho-MEK1/2), JAK2 (phospho-JAK2), and PTPN11 (phosho-PTPN11). Beta-actin and total protein signals were used as a loading control to normalize samples. (B) A blot separate from (A) demonstrates the TSLP-inducibility of RAS-effector bRAF. (C) RAS-GTP quantification of 5 independent ELISA experiments in which RAS activity of TSLP-induced MUTZ-5 cells was measured using a different, ELISA-specific active RAS pull-down assay. (D) TSLP-induced MUTZ-5 cells were probed for the presence of activated isoforms KRAS-GTP, HRAS-GTP, or NRAS-GTP (blots on the left). The blots on the right show the total expression of the respective RAS proteins and the graphs show the signal fold change over uninduced MUTZ-5 cells for KRAS-GTP, HRAS-GTP and NRAS-GTP of 4 independent experiments. All error bars are SD; P -values were calculated using Student’s T-test and are adjusted with a Bonferroni-correction for sequential multiple comparison.

    Journal: bioRxiv

    Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

    doi: 10.1101/2020.02.03.931725

    Figure Lengend Snippet: Human Ph-like B-ALL (spontaneous CRLF2-rearrangment and JAK2R683G mutation) cells activate wildtype RAS and RAS-interacting proteins upon TSLP induction. MUTZ-5 cells (Human Ph-like B-ALL cells bearing CRLF2-rearranged and spontaneous JAK2R683G mutation) were stimulated with 20 ng/mL human TSLP (maximal effective TSLP-concentration, Supplementary-Fig.S3B) for 10 min before cell lysis. Each cell lysate was split up for analysis in RAS-GTP pull-down assay and for total protein signal. (A) RAS-GTP pull-down and lysate samples were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, each membrane part was stripped and reprobed with new antibodies. RAS-GTP pull-down samples are on the left side while the right-hand side blots show whole cell lysates of the same samples. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band. The grey arrow shows the unspecific signal of the GST-RAS binding domain (RBD) used in the active RAS pull-down assay acting as a loading control. The experiment was repeated 5 times independently and the graphs show the quantification for active RAS (RAS-GTP), phosphorylated MEK1/2 (phospho-MEK1/2), JAK2 (phospho-JAK2), and PTPN11 (phosho-PTPN11). Beta-actin and total protein signals were used as a loading control to normalize samples. (B) A blot separate from (A) demonstrates the TSLP-inducibility of RAS-effector bRAF. (C) RAS-GTP quantification of 5 independent ELISA experiments in which RAS activity of TSLP-induced MUTZ-5 cells was measured using a different, ELISA-specific active RAS pull-down assay. (D) TSLP-induced MUTZ-5 cells were probed for the presence of activated isoforms KRAS-GTP, HRAS-GTP, or NRAS-GTP (blots on the left). The blots on the right show the total expression of the respective RAS proteins and the graphs show the signal fold change over uninduced MUTZ-5 cells for KRAS-GTP, HRAS-GTP and NRAS-GTP of 4 independent experiments. All error bars are SD; P -values were calculated using Student’s T-test and are adjusted with a Bonferroni-correction for sequential multiple comparison.

    Article Snippet: SDS-PAGE with 11%-resolving/5%-stacking acrylamide gels, and WB on PVDF-membrane (Cat.#88518; ThermoFisher Scientific) were performed according to the standard protocol of the equipment-manufacturer (Bio-Rad Laboratories).

    Techniques: Mutagenesis, Concentration Assay, Lysis, Pull Down Assay, SDS Page, Western Blot, Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing

    Direct wtRAS activation can precede PI3K/mTOR pathway activation, and the resulting PI3K downstream signaling activity can be blocked by RAS inhibitor. (A) Effect of TSLP induction over time. MUTZ-5 cells were incubated with 20 ng/mL human TSLP at 37 °C and 5% CO 2 for the indicated time points (0 min to 18 hrs) before cell lysis. Due to the centrifugation step of the suspension cells the TSLP is able to act for 5 min before lysis at timepoint 0. Each cell lysate was split up for analysis in RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down and lysate samples were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, membranes were stripped and reprobed with new antibodies. RAS-GTP pull-down elutions are on the left side while the right-hand side blots show whole cell lysates of the same samples. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band. (B) Activation of PI3K/mTOR downstream target rpS6 protein was monitored via PLA in high-throughput microscopy. MUTZ-5 cells were either not induced or induced with 20 ng/mL TSLP for 10 min. Where indicated, cells were pre-treated for 3 hrs with either DMSO (vehicle control), RAS inhibitor, or JAK inhibitor. Cells were fixed and permeabilized in a 96 well plate. After blocking, antibodies against phosphorylated rpS6 and total rpS6 were used in conjunction with PLA rabbit and mouse probes to allow specific readout of rpS6 activation in single cells in a high-throughput manner. Histograms show the distribution for a single experiment of the number of PLA spots in cells with at least 1 PLA spot (assay control is only shown in the bar graph). A minimum of 600 cells were analyzed per sample. Non-linear Gaussian fitting curves were plotted. Fluorescent microscope images show examples of PLA spots in MUTZ-5 cells for the respective treatment; white scale bars are 20 µm long. (C) The bar graph summarizes the average PLA spot counts of 3 independent experiments. Error bars are SD and P -values were determined in one-way ANOVA and post-hoc Bonferroni multiple comparison.

    Journal: bioRxiv

    Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

    doi: 10.1101/2020.02.03.931725

    Figure Lengend Snippet: Direct wtRAS activation can precede PI3K/mTOR pathway activation, and the resulting PI3K downstream signaling activity can be blocked by RAS inhibitor. (A) Effect of TSLP induction over time. MUTZ-5 cells were incubated with 20 ng/mL human TSLP at 37 °C and 5% CO 2 for the indicated time points (0 min to 18 hrs) before cell lysis. Due to the centrifugation step of the suspension cells the TSLP is able to act for 5 min before lysis at timepoint 0. Each cell lysate was split up for analysis in RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down and lysate samples were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, membranes were stripped and reprobed with new antibodies. RAS-GTP pull-down elutions are on the left side while the right-hand side blots show whole cell lysates of the same samples. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band. (B) Activation of PI3K/mTOR downstream target rpS6 protein was monitored via PLA in high-throughput microscopy. MUTZ-5 cells were either not induced or induced with 20 ng/mL TSLP for 10 min. Where indicated, cells were pre-treated for 3 hrs with either DMSO (vehicle control), RAS inhibitor, or JAK inhibitor. Cells were fixed and permeabilized in a 96 well plate. After blocking, antibodies against phosphorylated rpS6 and total rpS6 were used in conjunction with PLA rabbit and mouse probes to allow specific readout of rpS6 activation in single cells in a high-throughput manner. Histograms show the distribution for a single experiment of the number of PLA spots in cells with at least 1 PLA spot (assay control is only shown in the bar graph). A minimum of 600 cells were analyzed per sample. Non-linear Gaussian fitting curves were plotted. Fluorescent microscope images show examples of PLA spots in MUTZ-5 cells for the respective treatment; white scale bars are 20 µm long. (C) The bar graph summarizes the average PLA spot counts of 3 independent experiments. Error bars are SD and P -values were determined in one-way ANOVA and post-hoc Bonferroni multiple comparison.

    Article Snippet: SDS-PAGE with 11%-resolving/5%-stacking acrylamide gels, and WB on PVDF-membrane (Cat.#88518; ThermoFisher Scientific) were performed according to the standard protocol of the equipment-manufacturer (Bio-Rad Laboratories).

    Techniques: Activation Assay, Activity Assay, Incubation, Lysis, Centrifugation, Pull Down Assay, SDS Page, Western Blot, Labeling, Proximity Ligation Assay, High Throughput Screening Assay, Microscopy, Blocking Assay, Spot Test

    70% of primary bone marrow presentation samples of Down syndrome ALL patients show activated and/or TSLP-inducible RAS, regardless of mutations status. Primary presentation samples of DS-ALL patients were cultured for 2 days (see Supplementary-Fig.S4A legend for details) and then induced for 10 min with 20 ng/mL TSLP (or not induced) in serum-reduced medium. Lysates were analyzed for RAS activity in WB pull-down (A) or ELISA (B) using an ELISA-specific RAS pull-down assay. (A) Each lysate was split up for analysis in Western blot RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down (left) and lysate samples (right) were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, membranes were stripped and reprobed with new antibodies. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band; the grey arrow shows the loading of the GST-RBD in the pull-down assay. (B) The RAS activity pattern in the patient samples from (A) was confirmed via ELISA measurement of RAS-activity in aliquots that were independently thawed and processed as described above The line graph illustrates the four main patterns observed for RAS activity in primary ALL patient samples. (C) Shows an overview of the ELISA-measured RAS activity for the DS-ALL cohort at diagnosis (not enough cell material was available for DS26, DS29 and DS30). The RAS-GTP pull-down for ELISA was performed on lysates from cells at minimum 75% viability at a 100 ng/μL total protein concentration. In parallel, uninduced MUTZ-5 cells were subjected to the same treatment as the primary patient cells and were used to normalize all RAS activities. Brackets on top indicate the groups of the four RAS activity patterns presented in (A, B). For visualization purposes only in this graph, basal RAS activity over 50% of MUTZ-5 basal RAS activity was grouped as high RAS activity while an increase by at least 10% RAS-GTP in TSLP-stimulated samples over uninduced samples in ELISA was classed as TSLP-inducible RAS. For visualization purposes only in this graph, phosphorylation levels measured in WB for JAK2 were categorized as –(negative) = 0.00-0.05; + = 0.05-0.50; ++ = 0.50-1.00; +++ =1.00-2.00, and CRLF2 protein levels were categorized as –(negative) = 0.00-0.05; + = 0.05-0.20; ++ = 0.20-0.50; +++ =0.50-1.50. None of the arbitrary threshold groupings defined above were used in any of the PCA or clustering analysis shown later ( Fig.3 , Supplementary-FigS5). Known CRLF2-rearrangements are marked (R). All values are normalized to those measured for uninduced MUTZ-5 cells.). Outcome of leukemia is given (white = good outcome, black = poor outcome), and the presence of RAS mutations (blue) or JAK2 mutations (red) are specified (grey means unsequenced samples). The groups at the right end of the bar graph (separated by the black bar) show average RAS activities for patient/sample groups other than DS-ALL-diagnosis: Non-DS (NDS) at presentation, DS complete remission (CR) and DS/NDS at relapse. For an overview of the complete Western blot data and the quantified activities and protein expression of STAT5, JAK2, MEK1/2, ERK1/2 and rpS6 of all individual samples, see Supplementary-Fig.S4.

    Journal: bioRxiv

    Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

    doi: 10.1101/2020.02.03.931725

    Figure Lengend Snippet: 70% of primary bone marrow presentation samples of Down syndrome ALL patients show activated and/or TSLP-inducible RAS, regardless of mutations status. Primary presentation samples of DS-ALL patients were cultured for 2 days (see Supplementary-Fig.S4A legend for details) and then induced for 10 min with 20 ng/mL TSLP (or not induced) in serum-reduced medium. Lysates were analyzed for RAS activity in WB pull-down (A) or ELISA (B) using an ELISA-specific RAS pull-down assay. (A) Each lysate was split up for analysis in Western blot RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down (left) and lysate samples (right) were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, membranes were stripped and reprobed with new antibodies. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band; the grey arrow shows the loading of the GST-RBD in the pull-down assay. (B) The RAS activity pattern in the patient samples from (A) was confirmed via ELISA measurement of RAS-activity in aliquots that were independently thawed and processed as described above The line graph illustrates the four main patterns observed for RAS activity in primary ALL patient samples. (C) Shows an overview of the ELISA-measured RAS activity for the DS-ALL cohort at diagnosis (not enough cell material was available for DS26, DS29 and DS30). The RAS-GTP pull-down for ELISA was performed on lysates from cells at minimum 75% viability at a 100 ng/μL total protein concentration. In parallel, uninduced MUTZ-5 cells were subjected to the same treatment as the primary patient cells and were used to normalize all RAS activities. Brackets on top indicate the groups of the four RAS activity patterns presented in (A, B). For visualization purposes only in this graph, basal RAS activity over 50% of MUTZ-5 basal RAS activity was grouped as high RAS activity while an increase by at least 10% RAS-GTP in TSLP-stimulated samples over uninduced samples in ELISA was classed as TSLP-inducible RAS. For visualization purposes only in this graph, phosphorylation levels measured in WB for JAK2 were categorized as –(negative) = 0.00-0.05; + = 0.05-0.50; ++ = 0.50-1.00; +++ =1.00-2.00, and CRLF2 protein levels were categorized as –(negative) = 0.00-0.05; + = 0.05-0.20; ++ = 0.20-0.50; +++ =0.50-1.50. None of the arbitrary threshold groupings defined above were used in any of the PCA or clustering analysis shown later ( Fig.3 , Supplementary-FigS5). Known CRLF2-rearrangements are marked (R). All values are normalized to those measured for uninduced MUTZ-5 cells.). Outcome of leukemia is given (white = good outcome, black = poor outcome), and the presence of RAS mutations (blue) or JAK2 mutations (red) are specified (grey means unsequenced samples). The groups at the right end of the bar graph (separated by the black bar) show average RAS activities for patient/sample groups other than DS-ALL-diagnosis: Non-DS (NDS) at presentation, DS complete remission (CR) and DS/NDS at relapse. For an overview of the complete Western blot data and the quantified activities and protein expression of STAT5, JAK2, MEK1/2, ERK1/2 and rpS6 of all individual samples, see Supplementary-Fig.S4.

    Article Snippet: SDS-PAGE with 11%-resolving/5%-stacking acrylamide gels, and WB on PVDF-membrane (Cat.#88518; ThermoFisher Scientific) were performed according to the standard protocol of the equipment-manufacturer (Bio-Rad Laboratories).

    Techniques: Cell Culture, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Pull Down Assay, SDS Page, Labeling, Protein Concentration, Expressing

    Top Panel: SDS-PAGE analysis of time course digestion of Vip3Ab1 and Vip3Bc1 with H. zea and P. includens gut enzymes. Vip3Ab1 and Vip3Bc1 proteins (150 µg/mL) were incubated with gut fluids from H. zea (left) and P. includens (right) at 30 °C for various time intervals at pH 10.0. Bottom Panel: SDS-PAGE analysis of overnight digestion of Vip3 chimeras with H. zea gut enzymes. Vip3_AB and Vip3_BA proteins (110 µg/ml) were incubated with H. zea gut fluids for 16 hours at 30 °C in a total volume of 100 µL at pH 10.0. All reactions were stopped with protease inhibitors and 30 µL of the reaction loaded as described in Materials and Methods. Equivalent lanes were loaded and blotted onto a PVDF membrane for N-terminal sequencing.

    Journal: Scientific Reports

    Article Title: Functional characterization of Vip3Ab1 and Vip3Bc1: Two novel insecticidal proteins with differential activity against lepidopteran pests

    doi: 10.1038/s41598-017-11702-2

    Figure Lengend Snippet: Top Panel: SDS-PAGE analysis of time course digestion of Vip3Ab1 and Vip3Bc1 with H. zea and P. includens gut enzymes. Vip3Ab1 and Vip3Bc1 proteins (150 µg/mL) were incubated with gut fluids from H. zea (left) and P. includens (right) at 30 °C for various time intervals at pH 10.0. Bottom Panel: SDS-PAGE analysis of overnight digestion of Vip3 chimeras with H. zea gut enzymes. Vip3_AB and Vip3_BA proteins (110 µg/ml) were incubated with H. zea gut fluids for 16 hours at 30 °C in a total volume of 100 µL at pH 10.0. All reactions were stopped with protease inhibitors and 30 µL of the reaction loaded as described in Materials and Methods. Equivalent lanes were loaded and blotted onto a PVDF membrane for N-terminal sequencing.

    Article Snippet: Samples were prepared for N-terminal sequencing by blotting an SDS-PAGE gel onto a PVDF pre-cut blotting membrane (Thermo Scientific, Waltham, MA) via wet tank transfer in 10 mM CAPS (pH 11) with 10% methanol.

    Techniques: SDS Page, Incubation, Sequencing

    TNF-α induces formation of SDS-stable complexes between APP-βCTF and HS-anMan in growing mouse N2a neuroblastoma cells. The cells were either untreated (−) or grown for 48 h in the presence of 100 pg/ml of TNF-α (+). Immunoprecipitation (IP) was performed on the cell extracts with pAb A8717, which recognizes the C-terminus of APP. The same amount of protein was applied to each lane, and electrophoresis was followed by transfer to PVDF membranes and western blotting using either mAb 4G8 (for Aβ) or mAb AM (for HS-anMan). In lane 1, the primary antibody was omitted (0). A band corresponding to APP as in lane 4 was also seen when IP was performed with pAb Aβ40 (not shown).

    Journal: Glycobiology

    Article Title: Proinflammatory cytokines induce accumulation of glypican-1-derived heparan sulfate and the C-terminal fragment of β-cleaved APP in autophagosomes of dividing neuronal cells

    doi: 10.1093/glycob/cwaa011

    Figure Lengend Snippet: TNF-α induces formation of SDS-stable complexes between APP-βCTF and HS-anMan in growing mouse N2a neuroblastoma cells. The cells were either untreated (−) or grown for 48 h in the presence of 100 pg/ml of TNF-α (+). Immunoprecipitation (IP) was performed on the cell extracts with pAb A8717, which recognizes the C-terminus of APP. The same amount of protein was applied to each lane, and electrophoresis was followed by transfer to PVDF membranes and western blotting using either mAb 4G8 (for Aβ) or mAb AM (for HS-anMan). In lane 1, the primary antibody was omitted (0). A band corresponding to APP as in lane 4 was also seen when IP was performed with pAb Aβ40 (not shown).

    Article Snippet: Immunoisolates were displaced by using SDS, and SDS-PAGE was performed on 10% Tricine gels followed by transfer to polyvinylidene fluoride (PVDF) membranes, which were then probed with mAb 4G8 or mAb AM and visualized, all as described in detail previously ( ).

    Techniques: Immunoprecipitation, Electrophoresis, Western Blot