anti flag m2  (Millipore)


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    Structured Review

    Millipore anti flag m2
    Association of α4 with Mid1 and PP2A. ( a ) Yeast two-hybrid analysis showed that strains expressing Gal4-DBD-α4 grew on triple-drop out media ( trp -, leu -, his -) only when also expressing PP2A, PP6, or Mid1 (residues 58–180), indicative of an interaction with α4. Controls included single transformants and PP1C that did not support growth, because it does not bind α4. The plate was scanned on a densitometer to show growth of yeast colonies. ( b ) COS7 cells were transfected with <t>FLAG-tagged</t> Mid1(58–180) with or without myc -tagged α4. Cells were lysed and immunoprecipitation was performed by using <t>anti-FLAG</t> M2 affinity gel. FLAG-Mid1(58–180) and α4 were detected in the immunoprecipitates by immunoblotting with anti-FLAG and anti-α4 antibodies. When present, myc -α4 was detected by anti- myc immunoblotting. ( c ) COS7 cells transfected with vector alone, FLAG-α4 (full-length), FLAG-α4(1–249), FLAG-α4(220–340), or FLAG-α4(1–111) were lysed, and anti-FLAG immunoprecipitation was performed. FLAG-α4 coprecipitated endogenous Mid1 and PP2A-C, detected by anti-FLAG, anti-Mid1, and anti-PP2A-C immunoblotting.
    Anti Flag M2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit ?4"

    Article Title: Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit ?4

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

    doi: 10.1073/pnas.111154698

    Association of α4 with Mid1 and PP2A. ( a ) Yeast two-hybrid analysis showed that strains expressing Gal4-DBD-α4 grew on triple-drop out media ( trp -, leu -, his -) only when also expressing PP2A, PP6, or Mid1 (residues 58–180), indicative of an interaction with α4. Controls included single transformants and PP1C that did not support growth, because it does not bind α4. The plate was scanned on a densitometer to show growth of yeast colonies. ( b ) COS7 cells were transfected with FLAG-tagged Mid1(58–180) with or without myc -tagged α4. Cells were lysed and immunoprecipitation was performed by using anti-FLAG M2 affinity gel. FLAG-Mid1(58–180) and α4 were detected in the immunoprecipitates by immunoblotting with anti-FLAG and anti-α4 antibodies. When present, myc -α4 was detected by anti- myc immunoblotting. ( c ) COS7 cells transfected with vector alone, FLAG-α4 (full-length), FLAG-α4(1–249), FLAG-α4(220–340), or FLAG-α4(1–111) were lysed, and anti-FLAG immunoprecipitation was performed. FLAG-α4 coprecipitated endogenous Mid1 and PP2A-C, detected by anti-FLAG, anti-Mid1, and anti-PP2A-C immunoblotting.
    Figure Legend Snippet: Association of α4 with Mid1 and PP2A. ( a ) Yeast two-hybrid analysis showed that strains expressing Gal4-DBD-α4 grew on triple-drop out media ( trp -, leu -, his -) only when also expressing PP2A, PP6, or Mid1 (residues 58–180), indicative of an interaction with α4. Controls included single transformants and PP1C that did not support growth, because it does not bind α4. The plate was scanned on a densitometer to show growth of yeast colonies. ( b ) COS7 cells were transfected with FLAG-tagged Mid1(58–180) with or without myc -tagged α4. Cells were lysed and immunoprecipitation was performed by using anti-FLAG M2 affinity gel. FLAG-Mid1(58–180) and α4 were detected in the immunoprecipitates by immunoblotting with anti-FLAG and anti-α4 antibodies. When present, myc -α4 was detected by anti- myc immunoblotting. ( c ) COS7 cells transfected with vector alone, FLAG-α4 (full-length), FLAG-α4(1–249), FLAG-α4(220–340), or FLAG-α4(1–111) were lysed, and anti-FLAG immunoprecipitation was performed. FLAG-α4 coprecipitated endogenous Mid1 and PP2A-C, detected by anti-FLAG, anti-Mid1, and anti-PP2A-C immunoblotting.

    Techniques Used: Expressing, Transfection, Immunoprecipitation, Plasmid Preparation

    OS mutants of GFP-Mid1 associate with FLAG-α4. ( a ) COS7 cells were transfected to express GFP-Mid1(1–480) together with each of the following FLAG constructs: FLAG empty vector, FLAG-α4, and FLAG-α4(220–340). Cells were fixed and GFP-Mid1 was visualized by direct fluorescence microscopy. FLAG-α4 was visualized by anti-α4 immunofluorescence. Nuclei of the cells were visualized by DAPI staining. ( b ) COS7 cells were cotransfected to express FLAG-α4 and different GFP-Mid1 fusion constructs: GFP alone (as control), GFP-Mid1, GFP-Mid1(1–480), GFP-Mid1(L626P), and GFP-Mid1(C266R). Immunoprecipitation was performed by using anti-FLAG M2 affinity gel and FLAG-α4; GFP-Mid1 fusion proteins and PP2A were detected by immunoblotting with anti-FLAG, anti-GFP, and anti-PP2A. The mutant GFP-Mid1 proteins all bound FLAG-α4.
    Figure Legend Snippet: OS mutants of GFP-Mid1 associate with FLAG-α4. ( a ) COS7 cells were transfected to express GFP-Mid1(1–480) together with each of the following FLAG constructs: FLAG empty vector, FLAG-α4, and FLAG-α4(220–340). Cells were fixed and GFP-Mid1 was visualized by direct fluorescence microscopy. FLAG-α4 was visualized by anti-α4 immunofluorescence. Nuclei of the cells were visualized by DAPI staining. ( b ) COS7 cells were cotransfected to express FLAG-α4 and different GFP-Mid1 fusion constructs: GFP alone (as control), GFP-Mid1, GFP-Mid1(1–480), GFP-Mid1(L626P), and GFP-Mid1(C266R). Immunoprecipitation was performed by using anti-FLAG M2 affinity gel and FLAG-α4; GFP-Mid1 fusion proteins and PP2A were detected by immunoblotting with anti-FLAG, anti-GFP, and anti-PP2A. The mutant GFP-Mid1 proteins all bound FLAG-α4.

    Techniques Used: Transfection, Construct, Plasmid Preparation, Fluorescence, Microscopy, Immunofluorescence, Staining, Immunoprecipitation, Mutagenesis

    Dephosphorylation of GFP-Mid1 induced by α4 and MAP kinase activity required for Mid1 association with microtubules. ( a ) COS7 cells were cotransfected to express GFP-Mid1 together with either FLAG-α4 or FLAG empty vector. Immunoprecipitation was performed by using anti-FLAG M2 affinity gel, and FLAG-α4 and GFP-Mid1 were detected by anti-FLAG and anti-GFP antibodies. ( b ) COS7 cells were transfected as described and radiolabeled with [ 32 P]orthophosphate for 90 min. GFP-Mid1 was immunoprecipitated with anti-GFP antibodies and was subjected to PhosphorImager analysis after SDS/PAGE. Yield of GFP-Mid1 in the immunoprecipitates was determined by anti-GFP immunoblotting. FLAG-α4 in the cell lysate was detected by anti-FLAG immunoblotting. ( c ) COS7 cells were transfected to express GFP-Mid1, an initial image was captured (GFP:Mid1), and then the cells were treated with 10 μM UO126 and images were captured at the times indicated.
    Figure Legend Snippet: Dephosphorylation of GFP-Mid1 induced by α4 and MAP kinase activity required for Mid1 association with microtubules. ( a ) COS7 cells were cotransfected to express GFP-Mid1 together with either FLAG-α4 or FLAG empty vector. Immunoprecipitation was performed by using anti-FLAG M2 affinity gel, and FLAG-α4 and GFP-Mid1 were detected by anti-FLAG and anti-GFP antibodies. ( b ) COS7 cells were transfected as described and radiolabeled with [ 32 P]orthophosphate for 90 min. GFP-Mid1 was immunoprecipitated with anti-GFP antibodies and was subjected to PhosphorImager analysis after SDS/PAGE. Yield of GFP-Mid1 in the immunoprecipitates was determined by anti-GFP immunoblotting. FLAG-α4 in the cell lysate was detected by anti-FLAG immunoblotting. ( c ) COS7 cells were transfected to express GFP-Mid1, an initial image was captured (GFP:Mid1), and then the cells were treated with 10 μM UO126 and images were captured at the times indicated.

    Techniques Used: De-Phosphorylation Assay, Activity Assay, Plasmid Preparation, Immunoprecipitation, Transfection, SDS Page

    2) Product Images from "Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model"

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9388

    Histopathological analysis of tumors from Model #13 RetroLTR-EF and Model #14 piggyBac-EF A . Fibrosarcoma developed in Model #13 RetroLTR-EF . The storiform pattern of spindle-shaped, pleomorphic tumor cells is remarkable. Frequent mitotic figures (arrows) indicate aggressive tumor growth (left). α-SMA is a marker of smooth muscle and myofibroblastic cells. Human fibrosarcoma stains positive for α-SMA, whereas ES stains negative (middle). Expression of EWS-FLI1 in tumor tissue was confirmed using an anti-FLAG M2 antibody (right). B . Fibrosarcoma with a similar histology as (A) was also induced in Model #14 piggyBac-EF . Invasive growth of the tumor in lung tissue is noted (left). PDGF-RB is a mesenchymal marker that is frequently positive in human fibrosarcoma and negative in ES (middle). The expression of EWS-FLI1 was confirmed by western blotting (right).
    Figure Legend Snippet: Histopathological analysis of tumors from Model #13 RetroLTR-EF and Model #14 piggyBac-EF A . Fibrosarcoma developed in Model #13 RetroLTR-EF . The storiform pattern of spindle-shaped, pleomorphic tumor cells is remarkable. Frequent mitotic figures (arrows) indicate aggressive tumor growth (left). α-SMA is a marker of smooth muscle and myofibroblastic cells. Human fibrosarcoma stains positive for α-SMA, whereas ES stains negative (middle). Expression of EWS-FLI1 in tumor tissue was confirmed using an anti-FLAG M2 antibody (right). B . Fibrosarcoma with a similar histology as (A) was also induced in Model #14 piggyBac-EF . Invasive growth of the tumor in lung tissue is noted (left). PDGF-RB is a mesenchymal marker that is frequently positive in human fibrosarcoma and negative in ES (middle). The expression of EWS-FLI1 was confirmed by western blotting (right).

    Techniques Used: Marker, Expressing, Western Blot

    3) Product Images from "DNA display for in vitro selection of diverse peptide libraries"

    Article Title: DNA display for in vitro selection of diverse peptide libraries

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gng119

    Protein microarray analysis of randomly chosen peptides from rounds 3–5. Attachment of streptavidin-fused peptides on biotin-coated glass slides was confirmed with anti-T7·tag antibody (blue) and the peptides were screened with anti-FLAG M2 antibody (green). Streptavidin-fused FLAG peptide served as a positive control (red squares).
    Figure Legend Snippet: Protein microarray analysis of randomly chosen peptides from rounds 3–5. Attachment of streptavidin-fused peptides on biotin-coated glass slides was confirmed with anti-T7·tag antibody (blue) and the peptides were screened with anti-FLAG M2 antibody (green). Streptavidin-fused FLAG peptide served as a positive control (red squares).

    Techniques Used: Microarray, Positive Control

    Fraction of random decapeptide library that bound to anti-FLAG M2 antibody and was eluted with FLAG peptide, at each round of selection. The intensity of the selected peptide mixture relative to the original FLAG peptide is indicated.
    Figure Legend Snippet: Fraction of random decapeptide library that bound to anti-FLAG M2 antibody and was eluted with FLAG peptide, at each round of selection. The intensity of the selected peptide mixture relative to the original FLAG peptide is indicated.

    Techniques Used: Selection

    4) Product Images from "The Stability of Histone Acetyltransferase General Control Non-derepressible (Gcn) 5 Is Regulated by Cullin4-RING E3 Ubiquitin Ligase *"

    Article Title: The Stability of Histone Acetyltransferase General Control Non-derepressible (Gcn) 5 Is Regulated by Cullin4-RING E3 Ubiquitin Ligase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.290767

    And-1 prevents the ubiquitination of Gcn5 by CRL4 Cdt2 E3 ligase. A , overexpression of Cdt2 enhances the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested 48 h after transfection. Cells were treated with MG132 for 6 h before harvest. The ubiquitinated species were precipitated by HA antibodies and subjected to immunoblot for ubiquitinated FLAG-Gcn5 using anti-FLAG antibody. Ub , ubiquitin. B , overexpression of And-1 suppresses the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested for 48 h and treated as in A . Note that And-1 overexpression increased the FLAG-Gcn5 protein levels and reduced the ubiquitination of Gcn5. IP , immunoprecipitation; LC , loading control. C , And-1 prevents Gcn5 ubiquitination by CRL4 Cdt2 in vitro . Incubation of immunopurified CRL4 Cdt2 with Gcn5 in an in vitro ubiquitin ligase assay increased the formation of Gcn5 ubiquitinated species. The addition of immunopurified And-1 suppressed the Gcn5 ubiquitination by CRL4 Cdt2 . IF , immunofluorescence. D , And-1 overexpression suppresses the ubiquitination of the C terminus of Gcn5. U2OS cells transfected with the indicated plasmids were harvested. FLAG-And-1(1–491) or FLAG-And-1(492–837) was precipitated using anti-FLAG M2-agarose beads. FLAG precipitates and inputs were subjected to immunoblot using anti-ubiquitin, anti-FLAG, or anti-And-1 antibody.
    Figure Legend Snippet: And-1 prevents the ubiquitination of Gcn5 by CRL4 Cdt2 E3 ligase. A , overexpression of Cdt2 enhances the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested 48 h after transfection. Cells were treated with MG132 for 6 h before harvest. The ubiquitinated species were precipitated by HA antibodies and subjected to immunoblot for ubiquitinated FLAG-Gcn5 using anti-FLAG antibody. Ub , ubiquitin. B , overexpression of And-1 suppresses the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested for 48 h and treated as in A . Note that And-1 overexpression increased the FLAG-Gcn5 protein levels and reduced the ubiquitination of Gcn5. IP , immunoprecipitation; LC , loading control. C , And-1 prevents Gcn5 ubiquitination by CRL4 Cdt2 in vitro . Incubation of immunopurified CRL4 Cdt2 with Gcn5 in an in vitro ubiquitin ligase assay increased the formation of Gcn5 ubiquitinated species. The addition of immunopurified And-1 suppressed the Gcn5 ubiquitination by CRL4 Cdt2 . IF , immunofluorescence. D , And-1 overexpression suppresses the ubiquitination of the C terminus of Gcn5. U2OS cells transfected with the indicated plasmids were harvested. FLAG-And-1(1–491) or FLAG-And-1(492–837) was precipitated using anti-FLAG M2-agarose beads. FLAG precipitates and inputs were subjected to immunoblot using anti-ubiquitin, anti-FLAG, or anti-And-1 antibody.

    Techniques Used: Over Expression, Transfection, Immunoprecipitation, In Vitro, Incubation, Immunofluorescence

    Gcn5 forms complexes with CRL4 Cdt2 E3 ligase and And-1. A , Cdt2, DDB1, Cul4A, and And-1 interact with full-length ( Full ) Gcn5 and Gcn5 mutant ( 492–837 ). 293T cells transfected with the indicated FLAG-Gcn5 plasmids were harvested 36 h after transfection for immunoprecipitation using anti-FLAG M2-agarose beads. The FLAG immunoprecipitates ( IP ) were then subjected to immunoblot ( IB ) using anti-Cdt2, anti-Cul4A, anti-DDB1, anti-And-1, anti-actin, or anti-FLAG antibody. B , And-1 does not interact with Cdt2. 293T cells transfected with the indicated plasmids were treated as in A . Note that Gcn5 was detected in FLAG-And-1 precipitates.
    Figure Legend Snippet: Gcn5 forms complexes with CRL4 Cdt2 E3 ligase and And-1. A , Cdt2, DDB1, Cul4A, and And-1 interact with full-length ( Full ) Gcn5 and Gcn5 mutant ( 492–837 ). 293T cells transfected with the indicated FLAG-Gcn5 plasmids were harvested 36 h after transfection for immunoprecipitation using anti-FLAG M2-agarose beads. The FLAG immunoprecipitates ( IP ) were then subjected to immunoblot ( IB ) using anti-Cdt2, anti-Cul4A, anti-DDB1, anti-And-1, anti-actin, or anti-FLAG antibody. B , And-1 does not interact with Cdt2. 293T cells transfected with the indicated plasmids were treated as in A . Note that Gcn5 was detected in FLAG-And-1 precipitates.

    Techniques Used: Mutagenesis, Transfection, Immunoprecipitation

    And-1 competes with CRL4 Cdt2 for the association with Gcn5. A , schematic of Gcn5 truncation mutants used for protein-protein interactions as in B . The P300/CBP-associated factor (PCAF) homology domain ( PCAF-HD ), histone acetyltransferase ( HAT ) domain, and bromo domain ( BD ) are indicated. B , And-1 and CRL4 Cdt2 interact with Gcn5 at the same site within the C terminus. 293T cells transfected with the indicated plasmids were harvested and immunoprecipitated using anti-FLAG M2-agarose beads. FLAG-Gcn5 immunoprecipitates ( IP ) were subjected to immunoblot using anti-FLAG, anti-Cdt2, anti-DDB1, or anti-And-1 antibody. C , And-1 depletion increased the interaction between Gcn5 and CRL4 Cdt2 . U2OS cells transfected with the indicated siRNAs and FLAG-Gcn5 plasmid were treated with MG132 to stabilize Gcn5 before harvest. Cells were harvested 55 h after siRNA transfection for immunoprecipitation using anti-FLAG M2-agarose beads. FLAG-Gcn5 precipitates were subjected to immunoblot using anti-And-1, anti-Cdt2, anti-Cul4A, anti-actin, or anti-FLAG antibody. D , And-1 blocks the interaction between Cdt2 and Gcn5 in vitro . Immunopurified Gcn5 proteins on agarose beads were mixed with immunopurified FLAG-Cdt2 in the presence BSA or immunopurified FLAG-And-1 for 3 h at 4 °C. The beads were then washed three times using lysis buffer for immunoprecipitation. Gcn5 precipitates were subjected to immunoblot using anti-FLAG or anti-Gcn5 antibody.
    Figure Legend Snippet: And-1 competes with CRL4 Cdt2 for the association with Gcn5. A , schematic of Gcn5 truncation mutants used for protein-protein interactions as in B . The P300/CBP-associated factor (PCAF) homology domain ( PCAF-HD ), histone acetyltransferase ( HAT ) domain, and bromo domain ( BD ) are indicated. B , And-1 and CRL4 Cdt2 interact with Gcn5 at the same site within the C terminus. 293T cells transfected with the indicated plasmids were harvested and immunoprecipitated using anti-FLAG M2-agarose beads. FLAG-Gcn5 immunoprecipitates ( IP ) were subjected to immunoblot using anti-FLAG, anti-Cdt2, anti-DDB1, or anti-And-1 antibody. C , And-1 depletion increased the interaction between Gcn5 and CRL4 Cdt2 . U2OS cells transfected with the indicated siRNAs and FLAG-Gcn5 plasmid were treated with MG132 to stabilize Gcn5 before harvest. Cells were harvested 55 h after siRNA transfection for immunoprecipitation using anti-FLAG M2-agarose beads. FLAG-Gcn5 precipitates were subjected to immunoblot using anti-And-1, anti-Cdt2, anti-Cul4A, anti-actin, or anti-FLAG antibody. D , And-1 blocks the interaction between Cdt2 and Gcn5 in vitro . Immunopurified Gcn5 proteins on agarose beads were mixed with immunopurified FLAG-Cdt2 in the presence BSA or immunopurified FLAG-And-1 for 3 h at 4 °C. The beads were then washed three times using lysis buffer for immunoprecipitation. Gcn5 precipitates were subjected to immunoblot using anti-FLAG or anti-Gcn5 antibody.

    Techniques Used: HAT Assay, Transfection, Immunoprecipitation, Plasmid Preparation, In Vitro, Lysis

    5) Product Images from "Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified ?-Secretase Complexes"

    Article Title: Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified ?-Secretase Complexes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035133

    Enzymatic activity of partially purified γ-secretase complexes with FAD-linked PS1 mutants. (A) γ-Secretase activity assays performed with γ-MEF and γ - PS1/2 microsomal extracts prepared in 1% CHAPSO-HEPES buffer. Equal protein levels from the different extracts were diluted to 0.25% CHAPSO-HEPES buffer and incubated for 4 h at 37°C with lipids and 1 µM of recombinant human APP-based substrate (C100-Flag). Samples were analyzed by SDS-PAGE and immunostained with anti-Flag (M2) or anti-PS1 (MAB1563). The relative amounts of AICD-Flag generated in the reactions, reflecting γ-secretase activity, were estimated by densitometry. PS1 immunostaining was used to assess the amount of input material. (B) Equal amounts of microsomal proteins were immunoprecipitated overnight at 4°C with either anti-Flag M2 or anti-HA affinity resins, and submitted to a C100-His assay according to the same protocol as in (A). Protein samples were separated by SDS-PAGE and analysed by immunostaining for γ-secretase subunits ((NCT164 (NCT), MAB1563 (PS1-NTF), or UD1 (PEN2)). AICD-His cleavage products were immunostained with an anti-APP-CTF antibody (A8717). *Indicates a non-specific band corresponding to the IgG light chains. (C) Aβ1–40 and Aβ1–42 were quantified by sandwich ELISA and represented in pg/mL (left Y-axis) or in percentage (right Y-axis) of the mean of Aβ1–40 levels generated by the two wild-type clones. Aβ1–42/Aβ1–40 ratios are indicated on the top of the bars. The results were confirmed in three independent experiments and a representative dataset is shown.
    Figure Legend Snippet: Enzymatic activity of partially purified γ-secretase complexes with FAD-linked PS1 mutants. (A) γ-Secretase activity assays performed with γ-MEF and γ - PS1/2 microsomal extracts prepared in 1% CHAPSO-HEPES buffer. Equal protein levels from the different extracts were diluted to 0.25% CHAPSO-HEPES buffer and incubated for 4 h at 37°C with lipids and 1 µM of recombinant human APP-based substrate (C100-Flag). Samples were analyzed by SDS-PAGE and immunostained with anti-Flag (M2) or anti-PS1 (MAB1563). The relative amounts of AICD-Flag generated in the reactions, reflecting γ-secretase activity, were estimated by densitometry. PS1 immunostaining was used to assess the amount of input material. (B) Equal amounts of microsomal proteins were immunoprecipitated overnight at 4°C with either anti-Flag M2 or anti-HA affinity resins, and submitted to a C100-His assay according to the same protocol as in (A). Protein samples were separated by SDS-PAGE and analysed by immunostaining for γ-secretase subunits ((NCT164 (NCT), MAB1563 (PS1-NTF), or UD1 (PEN2)). AICD-His cleavage products were immunostained with an anti-APP-CTF antibody (A8717). *Indicates a non-specific band corresponding to the IgG light chains. (C) Aβ1–40 and Aβ1–42 were quantified by sandwich ELISA and represented in pg/mL (left Y-axis) or in percentage (right Y-axis) of the mean of Aβ1–40 levels generated by the two wild-type clones. Aβ1–42/Aβ1–40 ratios are indicated on the top of the bars. The results were confirmed in three independent experiments and a representative dataset is shown.

    Techniques Used: Activity Assay, Purification, Incubation, Recombinant, SDS Page, Generated, Immunostaining, Immunoprecipitation, Sandwich ELISA, Clone Assay

    6) Product Images from "Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection"

    Article Title: Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection

    Journal: Nature Communications

    doi: 10.1038/ncomms12628

    CtIP interacts with the CUL3-KLHL15 complex. ( a ) HEK293 cells inducibly expressing StrepHA-tagged CtIP were used for tandem affinity purification of protein complexes. The number of unique peptides and sequence coverage for the proteins identified by mass spectrometry are listed. ‘/' delimitates the data from two biological replicates. ( b ) HEK293T cells were transfected with either empty vector (EV) or the GFP-CtIP expression constructs. Forty-eight hours after transfection, cells were lysed and whole-cell extracts were subjected to IP using anti-GFP affinity resin. Inputs and recovered protein complexes were analysed by immunoblotting. ( c ) Schematic representation of the human KLHL15 protein indicating truncation and single-amino acid point mutants thereof used in d – f . ‘3-box' denotes CUL3-interacting box, whereas the six Kelch repeats are indicated in pink. ( d – f ) HEK293T cells were transfected with either EV or the indicated FLAG-KLHL15 expression constructs. Forty-eight hours after transfection, cells were lysed and whole-cell extracts were subjected to IP using anti-FLAG M2 affinity resin. Inputs and recovered protein complexes were analysed by immunoblotting. Asterisks indicate neddylated CUL3.
    Figure Legend Snippet: CtIP interacts with the CUL3-KLHL15 complex. ( a ) HEK293 cells inducibly expressing StrepHA-tagged CtIP were used for tandem affinity purification of protein complexes. The number of unique peptides and sequence coverage for the proteins identified by mass spectrometry are listed. ‘/' delimitates the data from two biological replicates. ( b ) HEK293T cells were transfected with either empty vector (EV) or the GFP-CtIP expression constructs. Forty-eight hours after transfection, cells were lysed and whole-cell extracts were subjected to IP using anti-GFP affinity resin. Inputs and recovered protein complexes were analysed by immunoblotting. ( c ) Schematic representation of the human KLHL15 protein indicating truncation and single-amino acid point mutants thereof used in d – f . ‘3-box' denotes CUL3-interacting box, whereas the six Kelch repeats are indicated in pink. ( d – f ) HEK293T cells were transfected with either EV or the indicated FLAG-KLHL15 expression constructs. Forty-eight hours after transfection, cells were lysed and whole-cell extracts were subjected to IP using anti-FLAG M2 affinity resin. Inputs and recovered protein complexes were analysed by immunoblotting. Asterisks indicate neddylated CUL3.

    Techniques Used: Expressing, Affinity Purification, Sequencing, Mass Spectrometry, Transfection, Plasmid Preparation, Construct

    7) Product Images from "SENP1-mediated deSUMOylation of USP28 regulated HIF-1α accumulation and activation during hypoxia response"

    Article Title: SENP1-mediated deSUMOylation of USP28 regulated HIF-1α accumulation and activation during hypoxia response

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0722-9

    Hypoxia enhanced the deubiquitinating activity of USP28 on HIF-1α. a HCT116 and A549 cells were cultured under normoxic (16% O 2 ) or hypoxic (5% O 2 ) conditions for 24 h. Cells were then lysed and USP28 protein levels were analyzed by western blot. The number on the bottom indicates signal intensity of USP28 protein against GAPDH. b 293T cells were transfected with Flag-tagged USP28 for 24 h and then cultured under normoxic or hypoxic conditions for additional 24 h. Then, Flag M2 beads were used to purify USP28 proteins in 293T cells. HA-HIF-1α, Myc-FBXW7 and His-Ubiquitin were co-transfected in 293T cells for 36 h. MG132 was added 4 h before cells were harvested. Ubiquitinated HIF-1α was purified from 293T cells using anti-HA antibody and used as the substrate of USP28 in vitro. BSA, normoxic USP28 or hypoxic USP28 proteins were added to ubiquitinated HIF-1α-FBXW7 complex for 1 h at 30 °C. 2Χ SDS loading buffer was then added to terminate the enzymatic reaction and the materials were subjected to western blot with indicated antibodies
    Figure Legend Snippet: Hypoxia enhanced the deubiquitinating activity of USP28 on HIF-1α. a HCT116 and A549 cells were cultured under normoxic (16% O 2 ) or hypoxic (5% O 2 ) conditions for 24 h. Cells were then lysed and USP28 protein levels were analyzed by western blot. The number on the bottom indicates signal intensity of USP28 protein against GAPDH. b 293T cells were transfected with Flag-tagged USP28 for 24 h and then cultured under normoxic or hypoxic conditions for additional 24 h. Then, Flag M2 beads were used to purify USP28 proteins in 293T cells. HA-HIF-1α, Myc-FBXW7 and His-Ubiquitin were co-transfected in 293T cells for 36 h. MG132 was added 4 h before cells were harvested. Ubiquitinated HIF-1α was purified from 293T cells using anti-HA antibody and used as the substrate of USP28 in vitro. BSA, normoxic USP28 or hypoxic USP28 proteins were added to ubiquitinated HIF-1α-FBXW7 complex for 1 h at 30 °C. 2Χ SDS loading buffer was then added to terminate the enzymatic reaction and the materials were subjected to western blot with indicated antibodies

    Techniques Used: Activity Assay, Cell Culture, Western Blot, Transfection, Purification, In Vitro

    SENP1 interacted with USP28 both in vivo and in vitro. a 293T cells were co-transfected with Flag-USP28 and GFP-SENP1 plasmids for 36 h. Cells were harvested and immunoprecipitation was performed with Flag M2 beads. The input and immunoprecipitates were subjected to western blot with indicated antibodies. b HCT116 cells were lysed and subjected to immunoprecipitation with mouse IgG or anti-USP28 antibody. The input and immunoprecipitates were subjected to western blot with indicated antibodies. c Bacterial purified GST-SENP1 proteins or GST alone were incubated with the purified Flag-USP28 protein for 1 h at 30 °C. Then the reaction was terminated by 2Χ SDS loading buffer and followed by western blot with indicated antibody
    Figure Legend Snippet: SENP1 interacted with USP28 both in vivo and in vitro. a 293T cells were co-transfected with Flag-USP28 and GFP-SENP1 plasmids for 36 h. Cells were harvested and immunoprecipitation was performed with Flag M2 beads. The input and immunoprecipitates were subjected to western blot with indicated antibodies. b HCT116 cells were lysed and subjected to immunoprecipitation with mouse IgG or anti-USP28 antibody. The input and immunoprecipitates were subjected to western blot with indicated antibodies. c Bacterial purified GST-SENP1 proteins or GST alone were incubated with the purified Flag-USP28 protein for 1 h at 30 °C. Then the reaction was terminated by 2Χ SDS loading buffer and followed by western blot with indicated antibody

    Techniques Used: In Vivo, In Vitro, Transfection, Immunoprecipitation, Western Blot, Purification, Incubation

    8) Product Images from "DNA Damage Induces the Accumulation of Tiam1 by Blocking β-TrCP-dependent Degradation *"

    Article Title: DNA Damage Induces the Accumulation of Tiam1 by Blocking β-TrCP-dependent Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.553388

    β-TrCP interacts with Tiam1 and controls its stability in a phosphorylation-dependent manner. A , cell lysates from HEK293T cells transfected with Myc-Tiam1 and FLAG-β-TrCP plasmids were subjected to IP with anti-FLAG M2 beads. WCL and
    Figure Legend Snippet: β-TrCP interacts with Tiam1 and controls its stability in a phosphorylation-dependent manner. A , cell lysates from HEK293T cells transfected with Myc-Tiam1 and FLAG-β-TrCP plasmids were subjected to IP with anti-FLAG M2 beads. WCL and

    Techniques Used: Transfection

    CK1 is involved in regulating Tiam1 stability. A , cell lysates from HEK293T cells transfected with Myc-Tiam1 and various FLAG-CK1 plasmids were subjected to IP with anti-FLAG M2 beads. WCL and immunoprecipitates were subjected to Western blot analysis
    Figure Legend Snippet: CK1 is involved in regulating Tiam1 stability. A , cell lysates from HEK293T cells transfected with Myc-Tiam1 and various FLAG-CK1 plasmids were subjected to IP with anti-FLAG M2 beads. WCL and immunoprecipitates were subjected to Western blot analysis

    Techniques Used: Transfection, Western Blot

    CK1 is required for β-TrCP/Tiam1 interaction. A , cell lysates from HEK293T cells transfected with Myc-Tiam1, FLAG-β-TrCP and Myc-dominant negative ( DN ) CK1δ plasmids were subjected to IP with anti-FLAG M2 beads. WCL and immunoprecipitates
    Figure Legend Snippet: CK1 is required for β-TrCP/Tiam1 interaction. A , cell lysates from HEK293T cells transfected with Myc-Tiam1, FLAG-β-TrCP and Myc-dominant negative ( DN ) CK1δ plasmids were subjected to IP with anti-FLAG M2 beads. WCL and immunoprecipitates

    Techniques Used: Transfection, Dominant Negative Mutation

    9) Product Images from "Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein"

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2013.10.015

    S114A, S167D, S200D, and S350D show cytoplasmic localization (A) HepG2 cells were transfected with the indicated PXR point mutant for 48 h and treated with either DMSO or 5 µM rifampicin (Rif) for 24 h. Cells were fixed, permeabilized, blocked, and then incubated with anti-Flag M2 antibody overnight. Fluorescence images were obtained at 40× magnification for detection of anti-Flag staining (green) and DAPI nuclear marker (blue). (B) The average fluorescence intensities of Flag-PXR in the nuclear and cytoplasmic compartments were calculated using the Cell Analysis Tool from Cellomics vHCS Toolbox, and the ratios of intensities (nuclear PXR/cytoplasmic PXR), represented on the y-axis, were compared with the PXR WT sample in each treatment group. Statistical analysis was performed using the Mann-Whitney non-parametric analysis, and * denotes statistical significance at p ≤ 0.05.
    Figure Legend Snippet: S114A, S167D, S200D, and S350D show cytoplasmic localization (A) HepG2 cells were transfected with the indicated PXR point mutant for 48 h and treated with either DMSO or 5 µM rifampicin (Rif) for 24 h. Cells were fixed, permeabilized, blocked, and then incubated with anti-Flag M2 antibody overnight. Fluorescence images were obtained at 40× magnification for detection of anti-Flag staining (green) and DAPI nuclear marker (blue). (B) The average fluorescence intensities of Flag-PXR in the nuclear and cytoplasmic compartments were calculated using the Cell Analysis Tool from Cellomics vHCS Toolbox, and the ratios of intensities (nuclear PXR/cytoplasmic PXR), represented on the y-axis, were compared with the PXR WT sample in each treatment group. Statistical analysis was performed using the Mann-Whitney non-parametric analysis, and * denotes statistical significance at p ≤ 0.05.

    Techniques Used: Transfection, Mutagenesis, Incubation, Fluorescence, Staining, Marker, MANN-WHITNEY

    10) Product Images from "A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis"

    Article Title: A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis

    Journal: Scientific Reports

    doi: 10.1038/srep11767

    Interaction of FLAG-BALF5 WT and 6A with EBV replication proteins. ( a and b ) HEK293EBV-BAC BALF5Δ cells were transfected with expression vectors for BZLF1 and FLAG-BALF5 wild type (WT) or 6A, with or without the expression vector for HA-tagged EBV replication proteins, as indicated. Cell proteins were lysed and incubated with (+) or without (−) Benzonase, then subjected to immunoprecipitation using anti-FLAG M2 antibody. The lysates (5% input) and precipitates (IP) were then immunoblotted using antibodies as indicated. ( c ) HEK293EBV-BAC BALF5Δ cells, transfected with expression vectors as noted (left of panels), were fixed and stained with anti-BALF2 (green), anti-FLAG (yellow) and anti-HA (red) antibodies, and observed by laser scanning confocal microscopy.
    Figure Legend Snippet: Interaction of FLAG-BALF5 WT and 6A with EBV replication proteins. ( a and b ) HEK293EBV-BAC BALF5Δ cells were transfected with expression vectors for BZLF1 and FLAG-BALF5 wild type (WT) or 6A, with or without the expression vector for HA-tagged EBV replication proteins, as indicated. Cell proteins were lysed and incubated with (+) or without (−) Benzonase, then subjected to immunoprecipitation using anti-FLAG M2 antibody. The lysates (5% input) and precipitates (IP) were then immunoblotted using antibodies as indicated. ( c ) HEK293EBV-BAC BALF5Δ cells, transfected with expression vectors as noted (left of panels), were fixed and stained with anti-BALF2 (green), anti-FLAG (yellow) and anti-HA (red) antibodies, and observed by laser scanning confocal microscopy.

    Techniques Used: BAC Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, Staining, Confocal Microscopy

    11) Product Images from "SENP1-mediated deSUMOylation of USP28 regulated HIF-1α accumulation and activation during hypoxia response"

    Article Title: SENP1-mediated deSUMOylation of USP28 regulated HIF-1α accumulation and activation during hypoxia response

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0722-9

    Hypoxia enhanced the deubiquitinating activity of USP28 on HIF-1α. a HCT116 and A549 cells were cultured under normoxic (16% O 2 ) or hypoxic (5% O 2 ) conditions for 24 h. Cells were then lysed and USP28 protein levels were analyzed by western blot. The number on the bottom indicates signal intensity of USP28 protein against GAPDH. b 293T cells were transfected with Flag-tagged USP28 for 24 h and then cultured under normoxic or hypoxic conditions for additional 24 h. Then, Flag M2 beads were used to purify USP28 proteins in 293T cells. HA-HIF-1α, Myc-FBXW7 and His-Ubiquitin were co-transfected in 293T cells for 36 h. MG132 was added 4 h before cells were harvested. Ubiquitinated HIF-1α was purified from 293T cells using anti-HA antibody and used as the substrate of USP28 in vitro. BSA, normoxic USP28 or hypoxic USP28 proteins were added to ubiquitinated HIF-1α-FBXW7 complex for 1 h at 30 °C. 2Χ SDS loading buffer was then added to terminate the enzymatic reaction and the materials were subjected to western blot with indicated antibodies
    Figure Legend Snippet: Hypoxia enhanced the deubiquitinating activity of USP28 on HIF-1α. a HCT116 and A549 cells were cultured under normoxic (16% O 2 ) or hypoxic (5% O 2 ) conditions for 24 h. Cells were then lysed and USP28 protein levels were analyzed by western blot. The number on the bottom indicates signal intensity of USP28 protein against GAPDH. b 293T cells were transfected with Flag-tagged USP28 for 24 h and then cultured under normoxic or hypoxic conditions for additional 24 h. Then, Flag M2 beads were used to purify USP28 proteins in 293T cells. HA-HIF-1α, Myc-FBXW7 and His-Ubiquitin were co-transfected in 293T cells for 36 h. MG132 was added 4 h before cells were harvested. Ubiquitinated HIF-1α was purified from 293T cells using anti-HA antibody and used as the substrate of USP28 in vitro. BSA, normoxic USP28 or hypoxic USP28 proteins were added to ubiquitinated HIF-1α-FBXW7 complex for 1 h at 30 °C. 2Χ SDS loading buffer was then added to terminate the enzymatic reaction and the materials were subjected to western blot with indicated antibodies

    Techniques Used: Activity Assay, Cell Culture, Western Blot, Transfection, Purification, In Vitro

    SENP1 interacted with USP28 both in vivo and in vitro. a 293T cells were co-transfected with Flag-USP28 and GFP-SENP1 plasmids for 36 h. Cells were harvested and immunoprecipitation was performed with Flag M2 beads. The input and immunoprecipitates were subjected to western blot with indicated antibodies. b HCT116 cells were lysed and subjected to immunoprecipitation with mouse IgG or anti-USP28 antibody. The input and immunoprecipitates were subjected to western blot with indicated antibodies. c Bacterial purified GST-SENP1 proteins or GST alone were incubated with the purified Flag-USP28 protein for 1 h at 30 °C. Then the reaction was terminated by 2Χ SDS loading buffer and followed by western blot with indicated antibody
    Figure Legend Snippet: SENP1 interacted with USP28 both in vivo and in vitro. a 293T cells were co-transfected with Flag-USP28 and GFP-SENP1 plasmids for 36 h. Cells were harvested and immunoprecipitation was performed with Flag M2 beads. The input and immunoprecipitates were subjected to western blot with indicated antibodies. b HCT116 cells were lysed and subjected to immunoprecipitation with mouse IgG or anti-USP28 antibody. The input and immunoprecipitates were subjected to western blot with indicated antibodies. c Bacterial purified GST-SENP1 proteins or GST alone were incubated with the purified Flag-USP28 protein for 1 h at 30 °C. Then the reaction was terminated by 2Χ SDS loading buffer and followed by western blot with indicated antibody

    Techniques Used: In Vivo, In Vitro, Transfection, Immunoprecipitation, Western Blot, Purification, Incubation

    12) Product Images from "The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor"

    Article Title: The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor

    Journal: Scientific Reports

    doi: 10.1038/srep28032

    Identification of the mutation in the patient sample and expression of the truncated FLT3 p.Q569Vfs*2 receptor. ( a ) Blast cells were isolated from the bone marrow of a relapsed AML patient. mRNA was isolated and reverse transcribed. The FLT3 cDNA was amplified and fragment analysis was performed. Arrows indicate fragments for FLT3 WT and FLT3 p.Q569Vfs*2. The peaks differ by eight base pairs in their fragment size. ( b ) Sanger sequencing revealed a deletion of eight nucleotides, leading to a frameshift and a premature stop codon within the FLT3 gene. The chromatogram is shown for the wild-type FLT3 , nucleotide triplets and the corresponding amino acids are shown for the FLT3 WT and the frameshift mutation FLT3 p.Q569Vfs*2 sequence. ( c ) Phoenix eco cells were transfected with FLAG-tagged FLT3 WT and FLAG-tagged FLT3 p.Q569Vfs*2. After cell lysis the FLT3 protein was immunoprecipitated from whole cell lysates with an N-terminal FLT3 antibody (SF1.340). After blotting the FLAG-tagged FLT3 was detected with an FLAG M2 antibody. One representative experiment is shown. The blot was cropped to improve the clarity of the image. ( d ) Ba/F3 cells stably expressing the indicated constructs. After cell lysis the FLT3 protein was detected with an N-terminal FLT3 antibody (4B12). One representative experiment is shown. FLT3 bands are indicated by asterisks. The blot was cropped to improve the clarity of the image (MT = FLT3 p.Q569Vfs*2). ( e ) Immunofluorescence of FLT3 (red), glycoconjugates (green) and counterstaining of DNA (blue) in transiently transfected U2OS cells. One representative image of each construct is shown.
    Figure Legend Snippet: Identification of the mutation in the patient sample and expression of the truncated FLT3 p.Q569Vfs*2 receptor. ( a ) Blast cells were isolated from the bone marrow of a relapsed AML patient. mRNA was isolated and reverse transcribed. The FLT3 cDNA was amplified and fragment analysis was performed. Arrows indicate fragments for FLT3 WT and FLT3 p.Q569Vfs*2. The peaks differ by eight base pairs in their fragment size. ( b ) Sanger sequencing revealed a deletion of eight nucleotides, leading to a frameshift and a premature stop codon within the FLT3 gene. The chromatogram is shown for the wild-type FLT3 , nucleotide triplets and the corresponding amino acids are shown for the FLT3 WT and the frameshift mutation FLT3 p.Q569Vfs*2 sequence. ( c ) Phoenix eco cells were transfected with FLAG-tagged FLT3 WT and FLAG-tagged FLT3 p.Q569Vfs*2. After cell lysis the FLT3 protein was immunoprecipitated from whole cell lysates with an N-terminal FLT3 antibody (SF1.340). After blotting the FLAG-tagged FLT3 was detected with an FLAG M2 antibody. One representative experiment is shown. The blot was cropped to improve the clarity of the image. ( d ) Ba/F3 cells stably expressing the indicated constructs. After cell lysis the FLT3 protein was detected with an N-terminal FLT3 antibody (4B12). One representative experiment is shown. FLT3 bands are indicated by asterisks. The blot was cropped to improve the clarity of the image (MT = FLT3 p.Q569Vfs*2). ( e ) Immunofluorescence of FLT3 (red), glycoconjugates (green) and counterstaining of DNA (blue) in transiently transfected U2OS cells. One representative image of each construct is shown.

    Techniques Used: Mutagenesis, Expressing, Isolation, Amplification, Sequencing, Transfection, Lysis, Immunoprecipitation, Stable Transfection, Construct, Immunofluorescence

    13) Product Images from "The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling"

    Article Title: The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021153

    PLIC-1 co-localized with Trif. The human hepatoma cells Huh7.5.1, were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 alone ( A ) or with 0.1 µg Flag-Trif ( B ) in a 24-well plate format. 24 hours post-infection, cells were fixed, permeabilized and stained with anti-Flag M2 antibody. Confocal and differential interference contrast (DIC) images were taken with a Zeiss Meta LSM510 microscope.  C . 0.1 µg of GFP expression plasmid was transfected with 0.1 µg Flag-Trif for confocal imaging.  D . 0.1 µg of YFP-PLIC-1 expression plasmid was transfected with 0.1 µg Flag-MAVS into Huh7.5.1 cells for confocal imaging.
    Figure Legend Snippet: PLIC-1 co-localized with Trif. The human hepatoma cells Huh7.5.1, were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 alone ( A ) or with 0.1 µg Flag-Trif ( B ) in a 24-well plate format. 24 hours post-infection, cells were fixed, permeabilized and stained with anti-Flag M2 antibody. Confocal and differential interference contrast (DIC) images were taken with a Zeiss Meta LSM510 microscope. C . 0.1 µg of GFP expression plasmid was transfected with 0.1 µg Flag-Trif for confocal imaging. D . 0.1 µg of YFP-PLIC-1 expression plasmid was transfected with 0.1 µg Flag-MAVS into Huh7.5.1 cells for confocal imaging.

    Techniques Used: Transfection, Infection, Staining, Microscopy, Expressing, Plasmid Preparation, Imaging

    14) Product Images from "β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation"

    Article Title: β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation

    Journal: Cancer Cell International

    doi: 10.1186/s12935-019-1029-1

    Dlg5 is associated with β-TrCP. a The interaction protein network of AEBP2 revealed by the BioGRID database. b 293T cell were transfected with Flag-Dlg5 and HA-β-TrCP for 36 h. Flag-Dlg5 protein complex was immunoprecipitated with Flag M2 beads. The Flag-DLG5 immunoprecipitate was detected by western blot using indicated antibodies. c SMMC-7721 cell lysate was subjected to immunoprecipitation by anti-Dlg5 antibody. Immunoprecipitates was detected by western blot using indicated antibodies
    Figure Legend Snippet: Dlg5 is associated with β-TrCP. a The interaction protein network of AEBP2 revealed by the BioGRID database. b 293T cell were transfected with Flag-Dlg5 and HA-β-TrCP for 36 h. Flag-Dlg5 protein complex was immunoprecipitated with Flag M2 beads. The Flag-DLG5 immunoprecipitate was detected by western blot using indicated antibodies. c SMMC-7721 cell lysate was subjected to immunoprecipitation by anti-Dlg5 antibody. Immunoprecipitates was detected by western blot using indicated antibodies

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    15) Product Images from "MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression"

    Article Title: MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-234

    MUC16 increases cFLIP expression to attenuate TRAIL-induced apoptosis. (A) Real-time PCR analysis of cFLIP S and cFLIP L transcript levels in Ctrl scFv and MUC16 scFv-expressing OVCAR3 cells. Results were standardized using primers of the housekeeping gene RPLPO. Data are expressed as fold change relative to levels observed in Ctrl scFv-expression OVCAR3 cells. Data are from two independent experiments. (B) Lysates from Ctrl scFv- and MUC16 scFv-expressing OVCAR3 cells were immunoblotted with cFLIP L and cFLIP S antibodies. (C) OVCAR3 Ctrl scFv and OVCAR3 MUC16 1:9#7 and 1:9#9 scFv were incubated with cycloheximide (100 μM). Lysate were obtained at different time points and immunoblot analysis was performed with anti-cFLIP L antibody. Intensity of cFLIP L signal was determined by densitometric scanning and plotted as the percentage of cFLIP L remaining compared with the control (0 h). (D) OVCAR3 Ctrl scFv and OVCAR3 MUC16 1:9#7 scFv were incubated with FLAG-tagged TRAIL (1 μg/ml) and the DISC was immunoprecipitated with anti-FLAG M2 antibody. The membrane was immunoblotted with anti-cFLIP L and with anti-cFLIP s antibodies. (E) OVCAR3 cells were transfected with control nontargeting siRNA (NT siRNA) or cFLIP siRNA pools and then incubated with TRAIL. Immunoblot analysis (24 h) was performed with anti-cFLIP L and anti-cFLIP S antibodies and showed decreased cFLIP L and cFLIP S expression in cFLIP siRNA-transfected cells. Apoptosis was qualitatively assessed by phase contrast microscopy in TRAIL treated cells (100 ng/ml; 6 h) and showed morphological changes suggestive of apoptotic cells in cFLIP siRNA-transfected cells. Representative fields from one of two independent experiments are shown. Apoptosis was also by ELISA as described in Methods and expressed as fold increased relative to control (untreated) cells with the mean of triplicates from three independent experiments ± SD. (F) Lysates from empty vector- and MUC16 CTD-expressing SKOV3 cells were immunoblotted with cFLIP L and cFLIP S antibodies.
    Figure Legend Snippet: MUC16 increases cFLIP expression to attenuate TRAIL-induced apoptosis. (A) Real-time PCR analysis of cFLIP S and cFLIP L transcript levels in Ctrl scFv and MUC16 scFv-expressing OVCAR3 cells. Results were standardized using primers of the housekeeping gene RPLPO. Data are expressed as fold change relative to levels observed in Ctrl scFv-expression OVCAR3 cells. Data are from two independent experiments. (B) Lysates from Ctrl scFv- and MUC16 scFv-expressing OVCAR3 cells were immunoblotted with cFLIP L and cFLIP S antibodies. (C) OVCAR3 Ctrl scFv and OVCAR3 MUC16 1:9#7 and 1:9#9 scFv were incubated with cycloheximide (100 μM). Lysate were obtained at different time points and immunoblot analysis was performed with anti-cFLIP L antibody. Intensity of cFLIP L signal was determined by densitometric scanning and plotted as the percentage of cFLIP L remaining compared with the control (0 h). (D) OVCAR3 Ctrl scFv and OVCAR3 MUC16 1:9#7 scFv were incubated with FLAG-tagged TRAIL (1 μg/ml) and the DISC was immunoprecipitated with anti-FLAG M2 antibody. The membrane was immunoblotted with anti-cFLIP L and with anti-cFLIP s antibodies. (E) OVCAR3 cells were transfected with control nontargeting siRNA (NT siRNA) or cFLIP siRNA pools and then incubated with TRAIL. Immunoblot analysis (24 h) was performed with anti-cFLIP L and anti-cFLIP S antibodies and showed decreased cFLIP L and cFLIP S expression in cFLIP siRNA-transfected cells. Apoptosis was qualitatively assessed by phase contrast microscopy in TRAIL treated cells (100 ng/ml; 6 h) and showed morphological changes suggestive of apoptotic cells in cFLIP siRNA-transfected cells. Representative fields from one of two independent experiments are shown. Apoptosis was also by ELISA as described in Methods and expressed as fold increased relative to control (untreated) cells with the mean of triplicates from three independent experiments ± SD. (F) Lysates from empty vector- and MUC16 CTD-expressing SKOV3 cells were immunoblotted with cFLIP L and cFLIP S antibodies.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Immunoprecipitation, Transfection, Microscopy, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

    Related Articles

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    Clone Assay:

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    Centrifugation:

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    Cycling Probe Technology:

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    Stable Transfection:

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    Synthesized:

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    Cytometry:

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    Blocking Assay:

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    End-sequence Profiling:

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    Real-time Polymerase Chain Reaction:

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    Microarray:

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    Incubation:

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    Expressing:

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    Article Snippet: Stable expression of the receptor was confirmed by real time-PCR, Western blot, immunoprecipitation and flow cytometry as described elsewhere . .. The following antibodies were used as recommended by the manufacturer: anti FLAG (M2), alpha-Tubulin (T6199), goat anti rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO, USA), FLT3 (SF1.340, S-18), phospho-tyrosine (PY99), goat anti mouse and goat anti rat secondary antibodies from Santa Cruz Biotechnology (CA, USA), ERK and phospho-ERK (Thr202/Tyr204) from Cell Signaling Technology (Danvers, MA, USA).

    Article Title: MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression
    Article Snippet: XTT, phenazine methosulfate, propidium iodide, cycloheximide, anti-Flag M2 and anti-tubulin were from Sigma (Oakville, ON, Canada). .. Anti-myc 9E10 antibody used for Western blot detection, real-time PCR Taqman Gene Expression Assay Master Mix, Flip primers for RT-PCR assays and Flip siRNA were from Life Technologies Inc (Burlington, ON, Canada).

    Modification:

    Article Title: DNA Damage Induces the Accumulation of Tiam1 by Blocking β-TrCP-dependent Degradation *
    Article Snippet: HEK293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium. .. Anti-FLAG M2, anti-FLAG M2-agarose, anti c-myc (rabbit)-agarose, propidium iodide, MG132, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, citric acid, thymidine, and CPT were purchased from Sigma-Aldrich.

    Article Title: A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis
    Article Snippet: Cell culture and reagents HEK293 and HEK293 EBV-bacterial artificial chromosome (BAC) BALF5Δ cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum. .. Anti-FLAG (M2), Anti-α/β-tubulin (#2148) and Anti-HA (6E2) antibodies were from Sigma and Cell Signaling, respectively.

    Western Blot:

    Article Title: SENP1-mediated deSUMOylation of USP28 regulated HIF-1α accumulation and activation during hypoxia response
    Article Snippet: .. Western blot assay was then performed by using the following antibodies: anti-USP28, anti-SENP1, anti-GST, anti-His, anti-GFP, anti-GAPDH (Santa Cruz, CA, USA), anti-HIF1a (H1alpha67, abcam), anti-Myc (Cell Signaling, Boston, MA, USA), anti-Flag M2 and anti-HA (Sigma, St Louis, MI, USA). .. Immunoprecipitation The immunoprecipitation process has been described previously with some modifications [ , ].

    Article Title: β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation
    Article Snippet: .. The primary antibodies used in western blotting included anti-Flag M2, anti-HA, anti-Ub, anti-Dlg5 (Sigma-aldrich); anti-β-TrCP (Cell signaling) and anti-Cullin1, anti-SKP1 and anti-β-actin (Santa Cruz). .. Immunoprecipitation (IP) Cells were lysed in lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5% Nonidet P40, Roche complete EDTA acid-free protease inhibitor cocktail) for 20 min at 4 °C.

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model
    Article Snippet: .. Expression of EWS-FLI1 was confirmed by western blotting using anti-FLAG M2 and anti-beta-tubulin antibodies (Sigma). .. Model #15 CreEP-TL-EF Generation of Ewsr1 fl/+:Fli1 fl/+ mice (C57BL/6) was described elsewhere [ ].

    Article Title: The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor
    Article Snippet: Stable expression of the receptor was confirmed by real time-PCR, Western blot, immunoprecipitation and flow cytometry as described elsewhere . .. The following antibodies were used as recommended by the manufacturer: anti FLAG (M2), alpha-Tubulin (T6199), goat anti rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO, USA), FLT3 (SF1.340, S-18), phospho-tyrosine (PY99), goat anti mouse and goat anti rat secondary antibodies from Santa Cruz Biotechnology (CA, USA), ERK and phospho-ERK (Thr202/Tyr204) from Cell Signaling Technology (Danvers, MA, USA).

    Article Title: MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression
    Article Snippet: Anti-FADD and anti-TRAIL R2 used to perform Western blotting were from EMD Millipore (Etobicoke, ON, Canada). .. XTT, phenazine methosulfate, propidium iodide, cycloheximide, anti-Flag M2 and anti-tubulin were from Sigma (Oakville, ON, Canada).

    Article Title: Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified ?-Secretase Complexes
    Article Snippet: Paragraph title: SDS-PAGE, Native PAGE, Western blotting and antibodies ... For immunoblot analysis, gels were transferred to nitrocellulose or PVDF membranes (Whatman), and probed with the following antibodies: anti-Nicastrin NCT164 (BD Bioscience), anti-V5-tag for NCT-V5 (Covance), MAB1563 (Millipore) or ab10281 (Abcam) for PS1 NTF, MAB5232 for PS1 CTF (Millipore), 3F10 for APH1aL-HA (Roche), anti-Flag M2 for Flag-PEN-2 or C100-Flag (Sigma-Aldrich), A8717 for APP CTF (Sigma-Aldrich), and A2066 for β-actin (Sigma-Aldrich).

    Transfection:

    Article Title: Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit ?4
    Article Snippet: .. After transfection, cells were washed with 1.2× PEM buffer (120 mM Pipes, pH 7.0/6 mM EGTA/2.4 mM MgCl2 ) and then fixed with 3.5% paraformaldehyde for 15 min. FLAG-α4 and microtubules were stained with anti-FLAG M2 and anti-β-tubulin antibodies plus a tetramethylrhodamine B isothiocyanate-labeled secondary antibody (Sigma). .. Nuclei were stained by using the DNA-specific stain 4′,6-diamidino-2-phenylindole (DAPI).

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model
    Article Snippet: EWS-FLI1 was also subcloned into piggyBac transposon plasmid (a gift from P. Liu), and BM-MSCs were transfected with PB-EF and transposase mRNA generated by using an mMESSAGE mMACHINE T7 Ultra kit (Ambion). .. Expression of EWS-FLI1 was confirmed by western blotting using anti-FLAG M2 and anti-beta-tubulin antibodies (Sigma).

    Protease Inhibitor:

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein
    Article Snippet: Rifampicin, EZ-View Red anti-Flag M2 affinity gel, histone H1 protein, Flag peptide, and anti-Flag M2 and anti-β-actin antibodies were obtained from Sigma-Aldrich (St. Louis, MO). .. Halt phosphatase inhibitor cocktail was obtained from Pierce Biotechnology (Rockford, IL) and protease inhibitor cocktail tablets were from Roche (Indianapolis, IN).

    Cell Culture:

    Article Title: DNA Damage Induces the Accumulation of Tiam1 by Blocking β-TrCP-dependent Degradation *
    Article Snippet: HEK293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium. .. Anti-FLAG M2, anti-FLAG M2-agarose, anti c-myc (rabbit)-agarose, propidium iodide, MG132, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, citric acid, thymidine, and CPT were purchased from Sigma-Aldrich.

    Article Title: A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis
    Article Snippet: Paragraph title: Cell culture and reagents ... Anti-FLAG (M2), Anti-α/β-tubulin (#2148) and Anti-HA (6E2) antibodies were from Sigma and Cell Signaling, respectively.

    Generated:

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model
    Article Snippet: EWS-FLI1 was also subcloned into piggyBac transposon plasmid (a gift from P. Liu), and BM-MSCs were transfected with PB-EF and transposase mRNA generated by using an mMESSAGE mMACHINE T7 Ultra kit (Ambion). .. Expression of EWS-FLI1 was confirmed by western blotting using anti-FLAG M2 and anti-beta-tubulin antibodies (Sigma).

    Imaging:

    Article Title: Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified ?-Secretase Complexes
    Article Snippet: For immunoblot analysis, gels were transferred to nitrocellulose or PVDF membranes (Whatman), and probed with the following antibodies: anti-Nicastrin NCT164 (BD Bioscience), anti-V5-tag for NCT-V5 (Covance), MAB1563 (Millipore) or ab10281 (Abcam) for PS1 NTF, MAB5232 for PS1 CTF (Millipore), 3F10 for APH1aL-HA (Roche), anti-Flag M2 for Flag-PEN-2 or C100-Flag (Sigma-Aldrich), A8717 for APP CTF (Sigma-Aldrich), and A2066 for β-actin (Sigma-Aldrich). .. The Odyssey infrared imaging system (LICOR) was used to detect the fluorescent signal.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression
    Article Snippet: XTT, phenazine methosulfate, propidium iodide, cycloheximide, anti-Flag M2 and anti-tubulin were from Sigma (Oakville, ON, Canada). .. Anti-myc 9E10 antibody used for Western blot detection, real-time PCR Taqman Gene Expression Assay Master Mix, Flip primers for RT-PCR assays and Flip siRNA were from Life Technologies Inc (Burlington, ON, Canada).

    Sonication:

    Article Title: Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection
    Article Snippet: Co-immunoprecipitation For FLAG and HA co-IP assays, cells were lysed in NP-40 extraction buffer, sonicated and clarified by centrifugation. .. A total of 1–3 mg of lysates were incubated with anti-FLAG M2 or anti-HA affinity resin (Sigma) for 2 h or overnight at 4 °C.

    Injection:

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model
    Article Snippet: The cells were transplanted subcutaneously or injected via tail vein into irradiated recipients. .. Expression of EWS-FLI1 was confirmed by western blotting using anti-FLAG M2 and anti-beta-tubulin antibodies (Sigma).

    Immunofluorescence:

    Article Title: The Stability of Histone Acetyltransferase General Control Non-derepressible (Gcn) 5 Is Regulated by Cullin4-RING E3 Ubiquitin Ligase *
    Article Snippet: Paragraph title: Antibodies and Immunofluorescence ... Anti-tubulin (T6199), anti-FLAG-M2, and anti-HA were from Sigma.

    Fluorescence:

    Article Title: Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit ?4
    Article Snippet: Paragraph title: Fluorescence Microscopy. ... After transfection, cells were washed with 1.2× PEM buffer (120 mM Pipes, pH 7.0/6 mM EGTA/2.4 mM MgCl2 ) and then fixed with 3.5% paraformaldehyde for 15 min. FLAG-α4 and microtubules were stained with anti-FLAG M2 and anti-β-tubulin antibodies plus a tetramethylrhodamine B isothiocyanate-labeled secondary antibody (Sigma).

    Flow Cytometry:

    Article Title: The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor
    Article Snippet: Stable expression of the receptor was confirmed by real time-PCR, Western blot, immunoprecipitation and flow cytometry as described elsewhere . .. The following antibodies were used as recommended by the manufacturer: anti FLAG (M2), alpha-Tubulin (T6199), goat anti rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO, USA), FLT3 (SF1.340, S-18), phospho-tyrosine (PY99), goat anti mouse and goat anti rat secondary antibodies from Santa Cruz Biotechnology (CA, USA), ERK and phospho-ERK (Thr202/Tyr204) from Cell Signaling Technology (Danvers, MA, USA).

    Article Title: MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression
    Article Snippet: TRAIL-Flag and anti-TRAIL-R1 to R4 receptor antibodies used for flow cytometry were from Alexis Biochemicals (San Diego, CA). .. XTT, phenazine methosulfate, propidium iodide, cycloheximide, anti-Flag M2 and anti-tubulin were from Sigma (Oakville, ON, Canada).

    Microscopy:

    Article Title: Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit ?4
    Article Snippet: Paragraph title: Fluorescence Microscopy. ... After transfection, cells were washed with 1.2× PEM buffer (120 mM Pipes, pH 7.0/6 mM EGTA/2.4 mM MgCl2 ) and then fixed with 3.5% paraformaldehyde for 15 min. FLAG-α4 and microtubules were stained with anti-FLAG M2 and anti-β-tubulin antibodies plus a tetramethylrhodamine B isothiocyanate-labeled secondary antibody (Sigma).

    Mouse Assay:

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model
    Article Snippet: BM-MSCs were then transduced with the EWS-FLI1 retrovirus and injected from tail vein into irradiated BALB/c mice (model #13). .. Expression of EWS-FLI1 was confirmed by western blotting using anti-FLAG M2 and anti-beta-tubulin antibodies (Sigma).

    Labeling:

    Article Title: A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis
    Article Snippet: Anti-FLAG (M2), Anti-α/β-tubulin (#2148) and Anti-HA (6E2) antibodies were from Sigma and Cell Signaling, respectively. .. Secondary goat anti-rabbit, anti-mouse, and anti-rat IgG antibodies conjugated with Alexa 488, 594, 680, and a Zenon mouse IgG labeling kit were obtained from Molecular Probes.

    Lysis:

    Article Title: SENP1-mediated deSUMOylation of USP28 regulated HIF-1α accumulation and activation during hypoxia response
    Article Snippet: Western blot Cells were harvested and lysed with cold lysis buffer (150 mM Tris–HCl, pH 6.8, 100 mM DTT, 2% SDS and 10% glycerol). .. Western blot assay was then performed by using the following antibodies: anti-USP28, anti-SENP1, anti-GST, anti-His, anti-GFP, anti-GAPDH (Santa Cruz, CA, USA), anti-HIF1a (H1alpha67, abcam), anti-Myc (Cell Signaling, Boston, MA, USA), anti-Flag M2 and anti-HA (Sigma, St Louis, MI, USA).

    Purification:

    Article Title: Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified ?-Secretase Complexes
    Article Snippet: Purified γ-secretase was resolved by electrophoresis on NativePAGE™ Novex® Bis-Tris 4–16% Gels for Blue Native (BN)-PAGE analysis (Invitrogen). .. For immunoblot analysis, gels were transferred to nitrocellulose or PVDF membranes (Whatman), and probed with the following antibodies: anti-Nicastrin NCT164 (BD Bioscience), anti-V5-tag for NCT-V5 (Covance), MAB1563 (Millipore) or ab10281 (Abcam) for PS1 NTF, MAB5232 for PS1 CTF (Millipore), 3F10 for APH1aL-HA (Roche), anti-Flag M2 for Flag-PEN-2 or C100-Flag (Sigma-Aldrich), A8717 for APP CTF (Sigma-Aldrich), and A2066 for β-actin (Sigma-Aldrich).

    SDS Page:

    Article Title: SENP1-mediated deSUMOylation of USP28 regulated HIF-1α accumulation and activation during hypoxia response
    Article Snippet: Proteins were separated by 10–12% SDS-PAGE, transferred to NC membranes. .. Western blot assay was then performed by using the following antibodies: anti-USP28, anti-SENP1, anti-GST, anti-His, anti-GFP, anti-GAPDH (Santa Cruz, CA, USA), anti-HIF1a (H1alpha67, abcam), anti-Myc (Cell Signaling, Boston, MA, USA), anti-Flag M2 and anti-HA (Sigma, St Louis, MI, USA).

    Article Title: β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation
    Article Snippet: The boiled samples were separated by 10% SDS–PAGE and transferred to nitrocellulose (NC) membranes (Whatman, GE Healthcare). .. The primary antibodies used in western blotting included anti-Flag M2, anti-HA, anti-Ub, anti-Dlg5 (Sigma-aldrich); anti-β-TrCP (Cell signaling) and anti-Cullin1, anti-SKP1 and anti-β-actin (Santa Cruz).

    Article Title: Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection
    Article Snippet: A total of 1–3 mg of lysates were incubated with anti-FLAG M2 or anti-HA affinity resin (Sigma) for 2 h or overnight at 4 °C. .. Beads were then washed four times with 1xTNE-T (50 mM Tris-HCl, pH 7.5, 140 mM NaCl, 5 mM EDTA and 1%Triton) and once with 1 × TNE (50 mM Tris-HCl, pH 7.5, 140 mM NaCl and 5 mM EDTA), complexes were boiled in SDS sample buffer and analysed by SDS–PAGE followed by immunoblotting.

    Article Title: Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified ?-Secretase Complexes
    Article Snippet: Paragraph title: SDS-PAGE, Native PAGE, Western blotting and antibodies ... For immunoblot analysis, gels were transferred to nitrocellulose or PVDF membranes (Whatman), and probed with the following antibodies: anti-Nicastrin NCT164 (BD Bioscience), anti-V5-tag for NCT-V5 (Covance), MAB1563 (Millipore) or ab10281 (Abcam) for PS1 NTF, MAB5232 for PS1 CTF (Millipore), 3F10 for APH1aL-HA (Roche), anti-Flag M2 for Flag-PEN-2 or C100-Flag (Sigma-Aldrich), A8717 for APP CTF (Sigma-Aldrich), and A2066 for β-actin (Sigma-Aldrich).

    Plasmid Preparation:

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model
    Article Snippet: EWS-FLI1 was also subcloned into piggyBac transposon plasmid (a gift from P. Liu), and BM-MSCs were transfected with PB-EF and transposase mRNA generated by using an mMESSAGE mMACHINE T7 Ultra kit (Ambion). .. Expression of EWS-FLI1 was confirmed by western blotting using anti-FLAG M2 and anti-beta-tubulin antibodies (Sigma).

    Article Title: The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor
    Article Snippet: The empty vector, MSCV-IRES-eGFP-FLT3 -WT, and MSCV-IRES-eGFP-FLT3 -W51 have been described previously . .. The following antibodies were used as recommended by the manufacturer: anti FLAG (M2), alpha-Tubulin (T6199), goat anti rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO, USA), FLT3 (SF1.340, S-18), phospho-tyrosine (PY99), goat anti mouse and goat anti rat secondary antibodies from Santa Cruz Biotechnology (CA, USA), ERK and phospho-ERK (Thr202/Tyr204) from Cell Signaling Technology (Danvers, MA, USA).

    Software:

    Article Title: Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit ?4
    Article Snippet: After transfection, cells were washed with 1.2× PEM buffer (120 mM Pipes, pH 7.0/6 mM EGTA/2.4 mM MgCl2 ) and then fixed with 3.5% paraformaldehyde for 15 min. FLAG-α4 and microtubules were stained with anti-FLAG M2 and anti-β-tubulin antibodies plus a tetramethylrhodamine B isothiocyanate-labeled secondary antibody (Sigma). .. Images were acquired on a Nikon Microphot-SA equipped with a Hamamatsu C4742 charge-coupled device camera driven by openlab 2.0.6 (Improvision, Lexington, MA) software.

    Irradiation:

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model
    Article Snippet: The cells were transplanted subcutaneously or injected via tail vein into irradiated recipients. .. Expression of EWS-FLI1 was confirmed by western blotting using anti-FLAG M2 and anti-beta-tubulin antibodies (Sigma).

    Co-Immunoprecipitation Assay:

    Article Title: Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection
    Article Snippet: Co-immunoprecipitation For FLAG and HA co-IP assays, cells were lysed in NP-40 extraction buffer, sonicated and clarified by centrifugation. .. A total of 1–3 mg of lysates were incubated with anti-FLAG M2 or anti-HA affinity resin (Sigma) for 2 h or overnight at 4 °C.

    In Vitro:

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model
    Article Snippet: Self-renewal activities of BM-MSCs were confirmed by in vitro clonogenic assays, and differentiation into multiple lineages were confirmed by induction toward osteogenic, chondrogenic, neuronal, adipogenic and myogenic differentiation [ ]. .. Expression of EWS-FLI1 was confirmed by western blotting using anti-FLAG M2 and anti-beta-tubulin antibodies (Sigma).

    Electrophoresis:

    Article Title: Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified ?-Secretase Complexes
    Article Snippet: Purified γ-secretase was resolved by electrophoresis on NativePAGE™ Novex® Bis-Tris 4–16% Gels for Blue Native (BN)-PAGE analysis (Invitrogen). .. For immunoblot analysis, gels were transferred to nitrocellulose or PVDF membranes (Whatman), and probed with the following antibodies: anti-Nicastrin NCT164 (BD Bioscience), anti-V5-tag for NCT-V5 (Covance), MAB1563 (Millipore) or ab10281 (Abcam) for PS1 NTF, MAB5232 for PS1 CTF (Millipore), 3F10 for APH1aL-HA (Roche), anti-Flag M2 for Flag-PEN-2 or C100-Flag (Sigma-Aldrich), A8717 for APP CTF (Sigma-Aldrich), and A2066 for β-actin (Sigma-Aldrich).

    Immunoprecipitation:

    Article Title: The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor
    Article Snippet: Stable expression of the receptor was confirmed by real time-PCR, Western blot, immunoprecipitation and flow cytometry as described elsewhere . .. The following antibodies were used as recommended by the manufacturer: anti FLAG (M2), alpha-Tubulin (T6199), goat anti rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO, USA), FLT3 (SF1.340, S-18), phospho-tyrosine (PY99), goat anti mouse and goat anti rat secondary antibodies from Santa Cruz Biotechnology (CA, USA), ERK and phospho-ERK (Thr202/Tyr204) from Cell Signaling Technology (Danvers, MA, USA).

    Article Title: MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression
    Article Snippet: XTT, phenazine methosulfate, propidium iodide, cycloheximide, anti-Flag M2 and anti-tubulin were from Sigma (Oakville, ON, Canada). .. Anti-Bax and anti-myc-789 used for immunoprecipitation experiments were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    BAC Assay:

    Article Title: A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis
    Article Snippet: Cell culture and reagents HEK293 and HEK293 EBV-bacterial artificial chromosome (BAC) BALF5Δ cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum. .. Anti-FLAG (M2), Anti-α/β-tubulin (#2148) and Anti-HA (6E2) antibodies were from Sigma and Cell Signaling, respectively.

    Staining:

    Article Title: Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit ?4
    Article Snippet: .. After transfection, cells were washed with 1.2× PEM buffer (120 mM Pipes, pH 7.0/6 mM EGTA/2.4 mM MgCl2 ) and then fixed with 3.5% paraformaldehyde for 15 min. FLAG-α4 and microtubules were stained with anti-FLAG M2 and anti-β-tubulin antibodies plus a tetramethylrhodamine B isothiocyanate-labeled secondary antibody (Sigma). .. Nuclei were stained by using the DNA-specific stain 4′,6-diamidino-2-phenylindole (DAPI).

    Clear Native PAGE:

    Article Title: Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified ?-Secretase Complexes
    Article Snippet: Paragraph title: SDS-PAGE, Native PAGE, Western blotting and antibodies ... For immunoblot analysis, gels were transferred to nitrocellulose or PVDF membranes (Whatman), and probed with the following antibodies: anti-Nicastrin NCT164 (BD Bioscience), anti-V5-tag for NCT-V5 (Covance), MAB1563 (Millipore) or ab10281 (Abcam) for PS1 NTF, MAB5232 for PS1 CTF (Millipore), 3F10 for APH1aL-HA (Roche), anti-Flag M2 for Flag-PEN-2 or C100-Flag (Sigma-Aldrich), A8717 for APP CTF (Sigma-Aldrich), and A2066 for β-actin (Sigma-Aldrich).

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  • 95
    Millipore monoclonal anti flag m2 antibody
    V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an <t>anti-c-Myc</t> antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with <t>anti-FLAG</t> antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 2407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 antibody/product/Millipore
    Average 95 stars, based on 2407 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag m2 antibody - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    95
    Millipore anti flag m2 affinity gel
    Drp1 binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, <t>FLAG-tagged</t> WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show <t>anti-FLAG</t> IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed GST, GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P
    Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1584 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
    Average 95 stars, based on 1584 article reviews
    Price from $9.99 to $1999.99
    anti flag m2 affinity gel - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    99
    Millipore anti flag m2
    Kaposin B binds to c-Myc for regulating endothelial cell angiogenic activities. a Nuclear distribution pattern of KSHV Kaposin B protein in cells. Immunofluorescence staining for Kaposin B proteins. HMEC1 cells with stable Kaposin B (KapB) expression were fixed, and Kaposin B proteins were detected using <t>anti-Flag</t> mAb, followed by anti–mouse IgG secondary antibody conjugated with FITC ( green ). Cell nuclei were counterstained with Hoechst 33342, while actin filaments with Texas Red phalloidin (Alexa Fluor 568). b HMEC1 cells stably expressing Kaposin B were subjected to the Transwell cell-migration assay ( n = 3). c Kaposin B increases cell motility in HUVEC. Primary HUVEC stably transduced with Kaposin B or the vector control by lentivirus were used for Transwell cell-migration assays ( n = 3). d Kaposin B enhances microvascular formation of HUVEC in an in vitro MatriGel angiogenesis assay. Pictures were taken after 6 h of incubation ( left ), and tube length was then measured and compared ( right ). e Schematic representation of miR-221/-222 proximal promoter. Three E-boxes (E1 and E2/3, in red ) were found. f Co-immunoprecipitation assays show Kaposin B and c-Myc form a protein complex. Cell lysates were prepared from HMEC1 cells stably expressing Kaposin B. Five micrograms of anti-FLAG (clone M2; left panel ), anti-HA ( right panel ) or isotype IgG control were incubated with 500 μl of cell extracts and then analyzed by western blotting with indicated mAbs. g The interaction between c-Myc and Kaposin B was independent of promoter DNA. Co-immunoprecipitation assays were performed with or without DNase pre-treatment on cell lysates prepared from HMEC1 cells stably expressing Kaposin B. Anti-FLAG (clone M2) or isotype IgG control were incubated with cell extracts, and pull-down products were analyzed by western blotting with anti-HA mAb for c-Myc (upper panel) or <t>anti-FLAG</t> M2 mAb for Kaposin B ( lower panel ). h - i Knockdown of endogenous c-Myc levels in Kaposin B(+) HUVECs inhibits Kaposin B-induced cell migration ( h , left ) and microvasculature formation ( i , right ) ( n = 3)
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    Image Search Results


    V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an anti-c-Myc antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with anti-FLAG antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an anti-c-Myc antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with anti-FLAG antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Recombinant, Immunoprecipitation, SDS Page, Western Blot, Negative Control

    Vacuum infiltration and purification of V H H9-sIgA. N. benthamiana leaves were vacuum infiltrated with a mixture of V H H9-Fc/SC/JC and p19. Tissue was collected at 12 dpi. Cell extracts were prepared under native conditions and separated with SDS-PAGE under non-reducing conditions. (A) Secretory IgA was purified with peptide M Agarose. Western blots were detected with anti-c-Myc antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1), tetrameric (No. 2, ∼176 kDa), trimeric (No. 3, ∼132 kDa), dimeric (No. 4, ∼88 kDa), and monomeric (No. 5, ∼44 kDa) V H H9-Fc. (B) Secretory IgA was purified with anti-FLAG agarose. Western blots were detected with anti-Flag antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1, ∼66 kDa), SC/trimeric V H H9-Fc/JC (No. 2, ∼206 kDa), SC/dimeric V H H9-Fc (No. 3, ∼160), SC/monomeric V H H9-Fc (No. 4, ∼110 kDa), and monomeric SC (No. 5). 10 μl of cell extract was loaded as a snapshot of the antibody produced in vivo .

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Vacuum infiltration and purification of V H H9-sIgA. N. benthamiana leaves were vacuum infiltrated with a mixture of V H H9-Fc/SC/JC and p19. Tissue was collected at 12 dpi. Cell extracts were prepared under native conditions and separated with SDS-PAGE under non-reducing conditions. (A) Secretory IgA was purified with peptide M Agarose. Western blots were detected with anti-c-Myc antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1), tetrameric (No. 2, ∼176 kDa), trimeric (No. 3, ∼132 kDa), dimeric (No. 4, ∼88 kDa), and monomeric (No. 5, ∼44 kDa) V H H9-Fc. (B) Secretory IgA was purified with anti-FLAG agarose. Western blots were detected with anti-Flag antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1, ∼66 kDa), SC/trimeric V H H9-Fc/JC (No. 2, ∼206 kDa), SC/dimeric V H H9-Fc (No. 3, ∼160), SC/monomeric V H H9-Fc (No. 4, ∼110 kDa), and monomeric SC (No. 5). 10 μl of cell extract was loaded as a snapshot of the antibody produced in vivo .

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Purification, SDS Page, Western Blot, Produced, In Vivo

    Binding of plant-produced V H H9-sIgA to EHEC O157:H7 intimin. (A) SPR binding of V H H9-sIgA purified using peptide M. Either plant-produced V H H9-sIgA (top) or E. coli -produced V H H9 monomer (bottom) was immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was flowed over the resulting surfaces at concentrations ranging from 0.3 to 5 nM. The experiment was conducted in duplicate. Black lines show data and red lines show fits. (B) ELISA binding of plant-produced V H H9-sIgA purified using either peptide M (left) or anti-FLAG antibody (right) and detected using either anti-bovine IgA antibody (top) or anti-FLAG antibody (bottom). Results are representative of two independent experiments.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Binding of plant-produced V H H9-sIgA to EHEC O157:H7 intimin. (A) SPR binding of V H H9-sIgA purified using peptide M. Either plant-produced V H H9-sIgA (top) or E. coli -produced V H H9 monomer (bottom) was immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was flowed over the resulting surfaces at concentrations ranging from 0.3 to 5 nM. The experiment was conducted in duplicate. Black lines show data and red lines show fits. (B) ELISA binding of plant-produced V H H9-sIgA purified using either peptide M (left) or anti-FLAG antibody (right) and detected using either anti-bovine IgA antibody (top) or anti-FLAG antibody (bottom). Results are representative of two independent experiments.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Binding Assay, Produced, SPR Assay, Purification, Enzyme-linked Immunosorbent Assay

    Design and production of individual subunits required for chimeric secretory IgA assembly. (A) Schematic of all produced subunits fully assembled into a chimeric antibody intended for secretory IgA functionality. It notably differs from the structure of native secretory IgA by the replacement of the Fab region with a camelid-derived variable heavy chain fragment (V H H). (B) Schematic representation of constructs used for Agrobacterium -mediated transient expression in N. benthamiana leaves. CaMV 35S, cauliflower mosaic virus 35S promoter; CPMV 5′UTR, 5′-untranslated region of Cowpea mosaic virus; PR1b, tobacco pathogenesis-related protein 1b signal peptide; V H Hx-Fc, fusion of a camelid-derived V H H to a bovine Fc where x is either 1, 3, 9, or 10, corresponding to the isolated V H Hs; SC, bovine secretory component; JC, bovine JC; c-Myc, FLAG, HA, detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide; CPMV 3′UTR, 3′-untranslated region of Cowpea mosaic virus; nos, nopaline synthase terminator sequence; the cassettes were cloned into pEAQ-DEST-1 plant expression vectors. Schematic not drawn to scale. Bold outlines indicate translated regions. (C) Monovalent affinities and kinetics of the interaction between V H Hs and MBP-Int277 by SPR (pH 7.4, 25°C). (D) Predicted protein size and number of glycosylation sites for each subunit. (E–G) Western blots of crude extract from leaves of N. benthamiana harvested at 6 dpi expressing V H H1, 3, 9, and 10-Fc along with p19, a suppressor of gene silencing (E) , SC (F) , and JC (G) . 10 μg of TSP was loaded in each lane.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Design and production of individual subunits required for chimeric secretory IgA assembly. (A) Schematic of all produced subunits fully assembled into a chimeric antibody intended for secretory IgA functionality. It notably differs from the structure of native secretory IgA by the replacement of the Fab region with a camelid-derived variable heavy chain fragment (V H H). (B) Schematic representation of constructs used for Agrobacterium -mediated transient expression in N. benthamiana leaves. CaMV 35S, cauliflower mosaic virus 35S promoter; CPMV 5′UTR, 5′-untranslated region of Cowpea mosaic virus; PR1b, tobacco pathogenesis-related protein 1b signal peptide; V H Hx-Fc, fusion of a camelid-derived V H H to a bovine Fc where x is either 1, 3, 9, or 10, corresponding to the isolated V H Hs; SC, bovine secretory component; JC, bovine JC; c-Myc, FLAG, HA, detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide; CPMV 3′UTR, 3′-untranslated region of Cowpea mosaic virus; nos, nopaline synthase terminator sequence; the cassettes were cloned into pEAQ-DEST-1 plant expression vectors. Schematic not drawn to scale. Bold outlines indicate translated regions. (C) Monovalent affinities and kinetics of the interaction between V H Hs and MBP-Int277 by SPR (pH 7.4, 25°C). (D) Predicted protein size and number of glycosylation sites for each subunit. (E–G) Western blots of crude extract from leaves of N. benthamiana harvested at 6 dpi expressing V H H1, 3, 9, and 10-Fc along with p19, a suppressor of gene silencing (E) , SC (F) , and JC (G) . 10 μg of TSP was loaded in each lane.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Produced, Derivative Assay, Construct, Expressing, Isolation, Sequencing, Clone Assay, SPR Assay, Western Blot

    Smad4 provides an example of a protein that must be methylated before it can be phosphorylated by GSK3 and translocated into MVBs by Wnt signaling. ( A ) Diagram of how FGF/EGF, Wnt, and TGF-β/BMP signaling cross-talk at the level of Smad4. MAPK/FGF (green) primes phosphorylation by GSK3 (blue) at three sites; the meArg site discovered in this study is shown in red. ( B ) Wnt addition for 20 min increased Smad4 methylation in transfected HEK-293T cells. S4-Flag and GAPDH serve as loading controls. ( C – E ) Phospho-Smad4-Flag relocalized to vesicular structures after 15 min of Wnt3a addition, but only in the absence of the competitive methylation inhibitor Adox. ( F ) A potential Smad4 arginine-methylation site was mutated (R272K) to prevent arginine methylation with minimal effect on the protein. ( G ) Smad4-Flag-WT immunoprecipitated from transfected HEK-293T lysates was recognized by asymmetric dimethyl-Arg antibody while the Smad4-Flag-R272K mutant was not. Thus, Smad4 contains a single meArg site. ( H ) Smad4 phosphorylation by GSK3 requires arginine methylation. Ratios under each lane and the merge panels indicate GSK3 phosphorylated Smad4/total Smad4-Flag. ( I – N ) In situ protease protection assay using digitonin and proteinase K showing that wild-type Smad4-Flag was translocated inside membrane-bound organelles when Wnt was added for 15 min ( J ) but digested when Triton X-100 was added ( K ). Smad4-R272K-Flag was not translocated into membrane vesicles and was degraded by proteinase K ( L and M ). Panels I′ – N′ show DAPI staining and differential interference contrast microscopy to visualize cellular contours in the corresponding cells shown above. ( O ) Smad4 wild type (WT), Smad4-R272K, and Smad4 mutated at the three GSK3 sites (phosphorylation-resistant Smad4-GM) were tested in TGF-β signaling assays. HaCaT cells permanently transfected with the CAGA12-luciferase reporter and constitutive CMV-Renilla (in which MAPK activation was primed by addition of EGF) were used. This indicates that in the context of TGF-β signaling arginine methylation is required for the integration of FGF, Wnt, and TGF-β signals. ** P

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

    Article Title: Arginine methylation is required for canonical Wnt signaling and endolysosomal trafficking

    doi: 10.1073/pnas.1804091115

    Figure Lengend Snippet: Smad4 provides an example of a protein that must be methylated before it can be phosphorylated by GSK3 and translocated into MVBs by Wnt signaling. ( A ) Diagram of how FGF/EGF, Wnt, and TGF-β/BMP signaling cross-talk at the level of Smad4. MAPK/FGF (green) primes phosphorylation by GSK3 (blue) at three sites; the meArg site discovered in this study is shown in red. ( B ) Wnt addition for 20 min increased Smad4 methylation in transfected HEK-293T cells. S4-Flag and GAPDH serve as loading controls. ( C – E ) Phospho-Smad4-Flag relocalized to vesicular structures after 15 min of Wnt3a addition, but only in the absence of the competitive methylation inhibitor Adox. ( F ) A potential Smad4 arginine-methylation site was mutated (R272K) to prevent arginine methylation with minimal effect on the protein. ( G ) Smad4-Flag-WT immunoprecipitated from transfected HEK-293T lysates was recognized by asymmetric dimethyl-Arg antibody while the Smad4-Flag-R272K mutant was not. Thus, Smad4 contains a single meArg site. ( H ) Smad4 phosphorylation by GSK3 requires arginine methylation. Ratios under each lane and the merge panels indicate GSK3 phosphorylated Smad4/total Smad4-Flag. ( I – N ) In situ protease protection assay using digitonin and proteinase K showing that wild-type Smad4-Flag was translocated inside membrane-bound organelles when Wnt was added for 15 min ( J ) but digested when Triton X-100 was added ( K ). Smad4-R272K-Flag was not translocated into membrane vesicles and was degraded by proteinase K ( L and M ). Panels I′ – N′ show DAPI staining and differential interference contrast microscopy to visualize cellular contours in the corresponding cells shown above. ( O ) Smad4 wild type (WT), Smad4-R272K, and Smad4 mutated at the three GSK3 sites (phosphorylation-resistant Smad4-GM) were tested in TGF-β signaling assays. HaCaT cells permanently transfected with the CAGA12-luciferase reporter and constitutive CMV-Renilla (in which MAPK activation was primed by addition of EGF) were used. This indicates that in the context of TGF-β signaling arginine methylation is required for the integration of FGF, Wnt, and TGF-β signals. ** P

    Article Snippet: Antibodies were obtained from the following sources: mouse monoclonal antibody against PRMT1 (Santa Cruz Biotechnology, sc-59648; 1:1,000 for immunostaining and Western blots); antibody against Lamp1 (Cell Signaling, 3243; 1:1,000 for immunostaining); antibodies against asymmetric dimethylarginine modifications (MilliporeSigma, ASYM24; 1:500 for immunostaining or Western blots); and (Abcam 21C7; 1:500 for immunostaining or Western blots); antibody against GSK3 (BD Biosciences, 610201; 1:500 for immunostaining and 1:1,000 for Western blots); antibody against K48-polyubiquitin (MilliporeSigma, 05–1307; 1:1,000 for immunostaining); antibody against Flag (MilliporeSigma, F1804; 1:5,000 for immunostaining or Western blots); antibody against GAPDH (Cell Signaling Technology, 2118; 1:1,000 for Western blots); and actin (MilliporeSigma, A2066; 1:1,000 for Western blots) were used as loading controls for Western blots.

    Techniques: Methylation, Transfection, Immunoprecipitation, Mutagenesis, In Situ, Staining, Microscopy, Luciferase, Activation Assay

    Drp1 binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed GST, GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P

    Journal: The Journal of Clinical Investigation

    Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice

    doi: 10.1172/JCI127277

    Figure Lengend Snippet: Drp1 binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed GST, GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P

    Article Snippet: Lysates were precleared with Protein A/G PLUS Beads (Santa Cruz Biotechnology) at 4°C for 30 minutes before IP with FLAG-M2 beads (anti-FLAG M2 Affinity Gel, MilliporeSigma, catalog A2220; RRID: AB_10063035) or anti-MFF1 antibody (Cell Signaling Technology, catalog 86668; RRID: AB_2734126) and protein A/G PLUS beads (Santa Cruz Biotechnology).

    Techniques: Cell Culture, Plasmid Preparation, Transfection, Purification, GST Pulldown Assay, Staining, SDS Page, Binding Assay, Mutagenesis, shRNA, Expressing, Stable Transfection, Sampling

    Actin cytoskeleton interacts with p-Drp1S600 and mitochondria. Cultured podocytes stably expressing FLAG-tagged versions of Drp1 (WT) and Drp1S600A were treated with HG (25 mM) for 48 hours. FLAG-tagged Drp1S600D was cultured under NG conditions. Cells were cross-linked, immunoprecipitated, and subjected to MS. KEGG Pathway analysis of protein interaction partners was performed. Top interaction pathways for Drp1S600 according to ( A ) the number of proteins and ( B ) enrichment score. ( C ) KEGG pathway analysis of the mutant proteins indicating the top pathways identified for each Drp1 mutant. ( D ) Podocytes stably expressing FLAG-tagged Drp1S600A or S600D in conjunction with untagged MFF or empty vector were immunoprecipitated with anti-FLAG agarose gel. The top 2 panels show recovery of MFF and Drp1 by immunoblotting (IB) following IP. Immunoblots in the bottom panels are for the WCL starting material. Different isoforms of MFF are indicated by a bracket on the right. ( E ) Podocytes were immunostained with Tomm20 (green) for mitochondria and rhodamine-phalloidin (red) for actin. From left to right, gray-scale image of mitochondria staining (green in merge), gray-scale images of actin staining (red in merge), and merged image. Scale bar: 25 μm. ( F ) Quantification of the overlap based on Mander’s coefficient for each condition. ( G ) Immunofluorescence staining of paraffin-embedded kidney sections. Sections were stained for total Drp1 (grayscale, green in merge) and Arp3 (grayscale, red in merge). Scale bar: 50 μm. ( H ) Colocalization analysis using Pearson’s correlation analysis of total Drp1 and mitochondria determined from the images represented in F . Representative images are from a sampling of 3 to5 separate cell cultures or animals. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice

    doi: 10.1172/JCI127277

    Figure Lengend Snippet: Actin cytoskeleton interacts with p-Drp1S600 and mitochondria. Cultured podocytes stably expressing FLAG-tagged versions of Drp1 (WT) and Drp1S600A were treated with HG (25 mM) for 48 hours. FLAG-tagged Drp1S600D was cultured under NG conditions. Cells were cross-linked, immunoprecipitated, and subjected to MS. KEGG Pathway analysis of protein interaction partners was performed. Top interaction pathways for Drp1S600 according to ( A ) the number of proteins and ( B ) enrichment score. ( C ) KEGG pathway analysis of the mutant proteins indicating the top pathways identified for each Drp1 mutant. ( D ) Podocytes stably expressing FLAG-tagged Drp1S600A or S600D in conjunction with untagged MFF or empty vector were immunoprecipitated with anti-FLAG agarose gel. The top 2 panels show recovery of MFF and Drp1 by immunoblotting (IB) following IP. Immunoblots in the bottom panels are for the WCL starting material. Different isoforms of MFF are indicated by a bracket on the right. ( E ) Podocytes were immunostained with Tomm20 (green) for mitochondria and rhodamine-phalloidin (red) for actin. From left to right, gray-scale image of mitochondria staining (green in merge), gray-scale images of actin staining (red in merge), and merged image. Scale bar: 25 μm. ( F ) Quantification of the overlap based on Mander’s coefficient for each condition. ( G ) Immunofluorescence staining of paraffin-embedded kidney sections. Sections were stained for total Drp1 (grayscale, green in merge) and Arp3 (grayscale, red in merge). Scale bar: 50 μm. ( H ) Colocalization analysis using Pearson’s correlation analysis of total Drp1 and mitochondria determined from the images represented in F . Representative images are from a sampling of 3 to5 separate cell cultures or animals. * P

    Article Snippet: Lysates were precleared with Protein A/G PLUS Beads (Santa Cruz Biotechnology) at 4°C for 30 minutes before IP with FLAG-M2 beads (anti-FLAG M2 Affinity Gel, MilliporeSigma, catalog A2220; RRID: AB_10063035) or anti-MFF1 antibody (Cell Signaling Technology, catalog 86668; RRID: AB_2734126) and protein A/G PLUS beads (Santa Cruz Biotechnology).

    Techniques: Cell Culture, Stable Transfection, Expressing, Immunoprecipitation, Mass Spectrometry, Mutagenesis, Plasmid Preparation, Agarose Gel Electrophoresis, Western Blot, Staining, Immunofluorescence, Sampling

    Kaposin B binds to c-Myc for regulating endothelial cell angiogenic activities. a Nuclear distribution pattern of KSHV Kaposin B protein in cells. Immunofluorescence staining for Kaposin B proteins. HMEC1 cells with stable Kaposin B (KapB) expression were fixed, and Kaposin B proteins were detected using anti-Flag mAb, followed by anti–mouse IgG secondary antibody conjugated with FITC ( green ). Cell nuclei were counterstained with Hoechst 33342, while actin filaments with Texas Red phalloidin (Alexa Fluor 568). b HMEC1 cells stably expressing Kaposin B were subjected to the Transwell cell-migration assay ( n = 3). c Kaposin B increases cell motility in HUVEC. Primary HUVEC stably transduced with Kaposin B or the vector control by lentivirus were used for Transwell cell-migration assays ( n = 3). d Kaposin B enhances microvascular formation of HUVEC in an in vitro MatriGel angiogenesis assay. Pictures were taken after 6 h of incubation ( left ), and tube length was then measured and compared ( right ). e Schematic representation of miR-221/-222 proximal promoter. Three E-boxes (E1 and E2/3, in red ) were found. f Co-immunoprecipitation assays show Kaposin B and c-Myc form a protein complex. Cell lysates were prepared from HMEC1 cells stably expressing Kaposin B. Five micrograms of anti-FLAG (clone M2; left panel ), anti-HA ( right panel ) or isotype IgG control were incubated with 500 μl of cell extracts and then analyzed by western blotting with indicated mAbs. g The interaction between c-Myc and Kaposin B was independent of promoter DNA. Co-immunoprecipitation assays were performed with or without DNase pre-treatment on cell lysates prepared from HMEC1 cells stably expressing Kaposin B. Anti-FLAG (clone M2) or isotype IgG control were incubated with cell extracts, and pull-down products were analyzed by western blotting with anti-HA mAb for c-Myc (upper panel) or anti-FLAG M2 mAb for Kaposin B ( lower panel ). h - i Knockdown of endogenous c-Myc levels in Kaposin B(+) HUVECs inhibits Kaposin B-induced cell migration ( h , left ) and microvasculature formation ( i , right ) ( n = 3)

    Journal: BMC Systems Biology

    Article Title: c-Myc and viral cofactor Kaposin B co-operate to elicit angiogenesis through modulating miRNome traits of endothelial cells

    doi: 10.1186/s12918-015-0242-3

    Figure Lengend Snippet: Kaposin B binds to c-Myc for regulating endothelial cell angiogenic activities. a Nuclear distribution pattern of KSHV Kaposin B protein in cells. Immunofluorescence staining for Kaposin B proteins. HMEC1 cells with stable Kaposin B (KapB) expression were fixed, and Kaposin B proteins were detected using anti-Flag mAb, followed by anti–mouse IgG secondary antibody conjugated with FITC ( green ). Cell nuclei were counterstained with Hoechst 33342, while actin filaments with Texas Red phalloidin (Alexa Fluor 568). b HMEC1 cells stably expressing Kaposin B were subjected to the Transwell cell-migration assay ( n = 3). c Kaposin B increases cell motility in HUVEC. Primary HUVEC stably transduced with Kaposin B or the vector control by lentivirus were used for Transwell cell-migration assays ( n = 3). d Kaposin B enhances microvascular formation of HUVEC in an in vitro MatriGel angiogenesis assay. Pictures were taken after 6 h of incubation ( left ), and tube length was then measured and compared ( right ). e Schematic representation of miR-221/-222 proximal promoter. Three E-boxes (E1 and E2/3, in red ) were found. f Co-immunoprecipitation assays show Kaposin B and c-Myc form a protein complex. Cell lysates were prepared from HMEC1 cells stably expressing Kaposin B. Five micrograms of anti-FLAG (clone M2; left panel ), anti-HA ( right panel ) or isotype IgG control were incubated with 500 μl of cell extracts and then analyzed by western blotting with indicated mAbs. g The interaction between c-Myc and Kaposin B was independent of promoter DNA. Co-immunoprecipitation assays were performed with or without DNase pre-treatment on cell lysates prepared from HMEC1 cells stably expressing Kaposin B. Anti-FLAG (clone M2) or isotype IgG control were incubated with cell extracts, and pull-down products were analyzed by western blotting with anti-HA mAb for c-Myc (upper panel) or anti-FLAG M2 mAb for Kaposin B ( lower panel ). h - i Knockdown of endogenous c-Myc levels in Kaposin B(+) HUVECs inhibits Kaposin B-induced cell migration ( h , left ) and microvasculature formation ( i , right ) ( n = 3)

    Article Snippet: The immunoprecipitates were probed with anti-FLAG M2 (1:2000) and anti-c-Myc Tag (clone 9E10; MILLIPORE, 05–419, 1:2000) monoclonal antibodies, and visualized with horseradish peroxidase-conjugated secondary antibodies using the ECL reagent (Amersham Pharmacia Biosciences).

    Techniques: Immunofluorescence, Staining, Expressing, Stable Transfection, Cell Migration Assay, Transduction, Plasmid Preparation, Migration, In Vitro, Angiogenesis Assay, Incubation, Immunoprecipitation, Western Blot