human htgl hek 293  (Millipore)


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    Structured Review

    Millipore human htgl hek 293
    Reactivity of MoAbs against recombinant <t>HTGL,</t> pre-HTGL, and PHP-HTGL by the immunoprecipitation (IP) method. Immunoprecipitated samples [1–10 (sample numbers are circled)] were separated by SDS-PAGE (5–20% gradient) under reducing condition. Following electrophoresis, the samples were subjected to immunoblot using 9A1 mouse MoAb (left) or 141A1 rat MoAb (right). Sample 1 is purified HTGL and samples 2–10 are fractions obtained by IP by the following method. Sample 2, IP of the culture supernatant of <t>HEK</t> 293 expressing HTGL with anti-LRG-48A1 mouse MoAb (negative control); sample 3, IP of the culture supernatant of HEK 293 expressing HTGL with anti-anti-HTGL-9A1 mouse MoAb; sample 4, IP of the culture supernatant of HEK 293 expressing HTGL with anti-HTGL-141A1 rat MoAb; sample 5, IP of serum with anti-LRG-48A1 mouse MoAb (negative control); sample 6, IP of serum with Anti-HTGL-9A1 mouse MoAb; sample 7, IP of serum with Anti-HTGL-141A1 rat MoAb; sample 8, IP of PHP with anti-LRG-48A1 mouse MoAb (negative control); sample 9, IP of PHP with anti-HTGL-9A1 mouse MoAb; sample 10, IP of PHP with anti-HTGL-141A1 rat MoAb. Multiple bands are shown in samples 3 and 4; we presume those are decomposed products from HTGL protein.
    Human Htgl Hek 293, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human htgl hek 293/product/Millipore
    Average 91 stars, based on 113 article reviews
    Price from $9.99 to $1999.99
    human htgl hek 293 - by Bioz Stars, 2020-09
    91/100 stars

    Images

    1) Product Images from "A new enzyme-linked immunosorbent assay system for human serum hepatic triglyceride lipase [S]"

    Article Title: A new enzyme-linked immunosorbent assay system for human serum hepatic triglyceride lipase [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M075432

    Reactivity of MoAbs against recombinant HTGL, pre-HTGL, and PHP-HTGL by the immunoprecipitation (IP) method. Immunoprecipitated samples [1–10 (sample numbers are circled)] were separated by SDS-PAGE (5–20% gradient) under reducing condition. Following electrophoresis, the samples were subjected to immunoblot using 9A1 mouse MoAb (left) or 141A1 rat MoAb (right). Sample 1 is purified HTGL and samples 2–10 are fractions obtained by IP by the following method. Sample 2, IP of the culture supernatant of HEK 293 expressing HTGL with anti-LRG-48A1 mouse MoAb (negative control); sample 3, IP of the culture supernatant of HEK 293 expressing HTGL with anti-anti-HTGL-9A1 mouse MoAb; sample 4, IP of the culture supernatant of HEK 293 expressing HTGL with anti-HTGL-141A1 rat MoAb; sample 5, IP of serum with anti-LRG-48A1 mouse MoAb (negative control); sample 6, IP of serum with Anti-HTGL-9A1 mouse MoAb; sample 7, IP of serum with Anti-HTGL-141A1 rat MoAb; sample 8, IP of PHP with anti-LRG-48A1 mouse MoAb (negative control); sample 9, IP of PHP with anti-HTGL-9A1 mouse MoAb; sample 10, IP of PHP with anti-HTGL-141A1 rat MoAb. Multiple bands are shown in samples 3 and 4; we presume those are decomposed products from HTGL protein.
    Figure Legend Snippet: Reactivity of MoAbs against recombinant HTGL, pre-HTGL, and PHP-HTGL by the immunoprecipitation (IP) method. Immunoprecipitated samples [1–10 (sample numbers are circled)] were separated by SDS-PAGE (5–20% gradient) under reducing condition. Following electrophoresis, the samples were subjected to immunoblot using 9A1 mouse MoAb (left) or 141A1 rat MoAb (right). Sample 1 is purified HTGL and samples 2–10 are fractions obtained by IP by the following method. Sample 2, IP of the culture supernatant of HEK 293 expressing HTGL with anti-LRG-48A1 mouse MoAb (negative control); sample 3, IP of the culture supernatant of HEK 293 expressing HTGL with anti-anti-HTGL-9A1 mouse MoAb; sample 4, IP of the culture supernatant of HEK 293 expressing HTGL with anti-HTGL-141A1 rat MoAb; sample 5, IP of serum with anti-LRG-48A1 mouse MoAb (negative control); sample 6, IP of serum with Anti-HTGL-9A1 mouse MoAb; sample 7, IP of serum with Anti-HTGL-141A1 rat MoAb; sample 8, IP of PHP with anti-LRG-48A1 mouse MoAb (negative control); sample 9, IP of PHP with anti-HTGL-9A1 mouse MoAb; sample 10, IP of PHP with anti-HTGL-141A1 rat MoAb. Multiple bands are shown in samples 3 and 4; we presume those are decomposed products from HTGL protein.

    Techniques Used: Recombinant, Immunoprecipitation, SDS Page, Electrophoresis, Purification, Expressing, Negative Control

    Sample purified from the concentrated conditioned medium of human HTGL/HEK 293 using an anti-FLAG (M2) monoclonal conjugated affinity gel was separated by SDS-PAGE (5–15% gradient) under reducing condition. Following electrophoresis, the sample was silver stained. Markers are shown at the left.
    Figure Legend Snippet: Sample purified from the concentrated conditioned medium of human HTGL/HEK 293 using an anti-FLAG (M2) monoclonal conjugated affinity gel was separated by SDS-PAGE (5–15% gradient) under reducing condition. Following electrophoresis, the sample was silver stained. Markers are shown at the left.

    Techniques Used: Purification, SDS Page, Electrophoresis, Staining

    2) Product Images from "ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with the Stat5b Pathway"

    Article Title: ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with the Stat5b Pathway

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E10-01-0040

    ZFP36L1 binding to AU-rich-elements (ARE) in the 3′UTR of Stat5b mRNA confers instability to mRNAs containing such elements. (A) Left, Western blot demonstrating identity and integrity of in vitro–translated FlagZFP36L1 protein: immunoblotting was performed with anti-Flag or anti-ZFP36L1 antibody as indicated. Right, RNA mobility shift assay performed by incubating in vitro–translated FlagZFP36L1 protein with labeled RNA probes corresponding to ARE in the 3′UTR of GM-CSF mRNA (used as a positive control, lanes 1–4) or to ARE in the 3′UTR of Stat5b mRNA (lanes 5–8). Probe sequences are: GM-CSF: 5′-UAUUUAUUUAUUUAUUUAUUUA-3′; Stat5b: 5′-AUAGUAAAUUAUUUAUUGGAAGAU-3′. Supershifts were obtained by an additional incubation with anti-Flag Ab (lanes 2 and 6). Competition experiments were performed with cold Stat5b probe (lane 3) or with cold GM-CSF probe (lane 7). Lanes 4 and 8 represent labeled probes incubated with an in vitro translation reaction mix performed on empty vector. (B) Luciferase activity assay performed in HEK293 cells transfected with pcDNA3.1 empty expression vector or with pcDNA3.1 overexpressing FlagZFP36L1 (20 ng) together with pGL3 reporter construct encoding for a luciferase gene fused to the 3′UTR of Vegfa (positive control) or to the 3′UTR of Stat5b. Luciferase activity is represented in terms of fold change; error bars, SEM calculated on a set of five independent experiments. (C) Ribonucleoprotein complexes immunoprecipitation assay demonstrating that binding of ZFP36L1 protein to the ARE in the 3′UTR of Stat5b mRNA occurs in vivo: ribonucleoprotein complexes were immunoprecipitated from lysates of HEK293 cells transfected with pcDNA3.1 ZFP36L1 and pGL3 3′UTR Stat5b vectors, RNA was extracted, reverse-transcribed, and amplified by PCR using primers specific for Stat5b 3′UTR fused to the luciferase gene. Lanes 1 and 2, negative and positive control respectively, i.e., PCR amplification performed on no template or on RNA extracted from cell lysates before immunoprecipitation; lane 3, specific immunoprecipitation obtained with anti-ZFP36L1 antibody; lanes 4 and 5, control immunoprecipitations performed with nonspecific antibody or with no antibody.
    Figure Legend Snippet: ZFP36L1 binding to AU-rich-elements (ARE) in the 3′UTR of Stat5b mRNA confers instability to mRNAs containing such elements. (A) Left, Western blot demonstrating identity and integrity of in vitro–translated FlagZFP36L1 protein: immunoblotting was performed with anti-Flag or anti-ZFP36L1 antibody as indicated. Right, RNA mobility shift assay performed by incubating in vitro–translated FlagZFP36L1 protein with labeled RNA probes corresponding to ARE in the 3′UTR of GM-CSF mRNA (used as a positive control, lanes 1–4) or to ARE in the 3′UTR of Stat5b mRNA (lanes 5–8). Probe sequences are: GM-CSF: 5′-UAUUUAUUUAUUUAUUUAUUUA-3′; Stat5b: 5′-AUAGUAAAUUAUUUAUUGGAAGAU-3′. Supershifts were obtained by an additional incubation with anti-Flag Ab (lanes 2 and 6). Competition experiments were performed with cold Stat5b probe (lane 3) or with cold GM-CSF probe (lane 7). Lanes 4 and 8 represent labeled probes incubated with an in vitro translation reaction mix performed on empty vector. (B) Luciferase activity assay performed in HEK293 cells transfected with pcDNA3.1 empty expression vector or with pcDNA3.1 overexpressing FlagZFP36L1 (20 ng) together with pGL3 reporter construct encoding for a luciferase gene fused to the 3′UTR of Vegfa (positive control) or to the 3′UTR of Stat5b. Luciferase activity is represented in terms of fold change; error bars, SEM calculated on a set of five independent experiments. (C) Ribonucleoprotein complexes immunoprecipitation assay demonstrating that binding of ZFP36L1 protein to the ARE in the 3′UTR of Stat5b mRNA occurs in vivo: ribonucleoprotein complexes were immunoprecipitated from lysates of HEK293 cells transfected with pcDNA3.1 ZFP36L1 and pGL3 3′UTR Stat5b vectors, RNA was extracted, reverse-transcribed, and amplified by PCR using primers specific for Stat5b 3′UTR fused to the luciferase gene. Lanes 1 and 2, negative and positive control respectively, i.e., PCR amplification performed on no template or on RNA extracted from cell lysates before immunoprecipitation; lane 3, specific immunoprecipitation obtained with anti-ZFP36L1 antibody; lanes 4 and 5, control immunoprecipitations performed with nonspecific antibody or with no antibody.

    Techniques Used: Binding Assay, Western Blot, In Vitro, Mobility Shift, Labeling, Positive Control, Incubation, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Construct, Immunoprecipitation, In Vivo, Amplification, Polymerase Chain Reaction

    ZFP36 behaves similarly to ZFP36L1 in binding to and destabilizing mRNAs spanning Stat5b 3′UTR and in inhibiting erythroid differentiation of CD34+ HSCs. (A) Left, Western blot demonstrating identity and integrity of in vitro–translated FlagZFP36 protein: immunoblotting was performed with anti-Flag or anti-ZFP36 antibody as indicated. Right, RNA mobility shift assay performed by incubating in vitro–translated FlagZFP36 protein with labeled RNA probes spanning the ARE in the 3′UTR of GM-CSF mRNA (used as a positive control, lanes 1–4) or the ARE in the 3′UTR of Stat5b mRNA (lanes 5–8). Supershifts were obtained by an additional incubation with anti-Flag Ab (lanes 2 and 6). Competition experiments were performed with cold Stat5b probe (lane 3) or with cold GM-CSF probe (lane 7). Lanes 4 and 8 represent labeled probes incubated with an in vitro translation reaction mix performed on empty vector. (B) Luciferase activity assay performed in HEK293 cells transfected with pcDNA3.1 empty expression vector (left) or with pcDNA3.1 overexpressing FlagZFP36 (10 ng; right) together with pGL3 reporter construct encoding for a luciferase gene fused to the 3′UTR of Stat5b. Luciferase activity is represented in terms of fold change; error bars, SEM calculated on a set of five independent experiments. (C) Clonogenic assay performed on CD34+ HSCs transduced with empty vector (left) or overexpressing ZFP36 (right); error bars, SEM calculated on four independent experiments (*p
    Figure Legend Snippet: ZFP36 behaves similarly to ZFP36L1 in binding to and destabilizing mRNAs spanning Stat5b 3′UTR and in inhibiting erythroid differentiation of CD34+ HSCs. (A) Left, Western blot demonstrating identity and integrity of in vitro–translated FlagZFP36 protein: immunoblotting was performed with anti-Flag or anti-ZFP36 antibody as indicated. Right, RNA mobility shift assay performed by incubating in vitro–translated FlagZFP36 protein with labeled RNA probes spanning the ARE in the 3′UTR of GM-CSF mRNA (used as a positive control, lanes 1–4) or the ARE in the 3′UTR of Stat5b mRNA (lanes 5–8). Supershifts were obtained by an additional incubation with anti-Flag Ab (lanes 2 and 6). Competition experiments were performed with cold Stat5b probe (lane 3) or with cold GM-CSF probe (lane 7). Lanes 4 and 8 represent labeled probes incubated with an in vitro translation reaction mix performed on empty vector. (B) Luciferase activity assay performed in HEK293 cells transfected with pcDNA3.1 empty expression vector (left) or with pcDNA3.1 overexpressing FlagZFP36 (10 ng; right) together with pGL3 reporter construct encoding for a luciferase gene fused to the 3′UTR of Stat5b. Luciferase activity is represented in terms of fold change; error bars, SEM calculated on a set of five independent experiments. (C) Clonogenic assay performed on CD34+ HSCs transduced with empty vector (left) or overexpressing ZFP36 (right); error bars, SEM calculated on four independent experiments (*p

    Techniques Used: Binding Assay, Western Blot, In Vitro, Mobility Shift, Labeling, Positive Control, Incubation, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Construct, Clonogenic Assay, Transduction

    3) Product Images from "The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis"

    Article Title: The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2001.01622.x

    Flow cytometric profiles, Western and Northern blotting of the FG1 and FG3 transfected cells. (a) Typical flow cytometric histograms for the FG1 and FG3 transfectants are shown. The control cells and stable transfectants with FG1 or FG3 were treated with anti-FLAG antibody (thick line) or isotype antibody (thin line). The mean shift values of each cell for anti-FLAG antibody and isotype antibody (inside parethesis) are indicated in each panel. (b) Western blotting of the transfectants. A whole cell lysate was separated by SDS-PAGE and Western blotting with anti-FLAG MoAb is shown. (c) Northern blottings of the transfectants. The probe used for the hybridization was the cDNA restriction fragment of HLA-G α1 domain.
    Figure Legend Snippet: Flow cytometric profiles, Western and Northern blotting of the FG1 and FG3 transfected cells. (a) Typical flow cytometric histograms for the FG1 and FG3 transfectants are shown. The control cells and stable transfectants with FG1 or FG3 were treated with anti-FLAG antibody (thick line) or isotype antibody (thin line). The mean shift values of each cell for anti-FLAG antibody and isotype antibody (inside parethesis) are indicated in each panel. (b) Western blotting of the transfectants. A whole cell lysate was separated by SDS-PAGE and Western blotting with anti-FLAG MoAb is shown. (c) Northern blottings of the transfectants. The probe used for the hybridization was the cDNA restriction fragment of HLA-G α1 domain.

    Techniques Used: Flow Cytometry, Western Blot, Northern Blot, Transfection, SDS Page, Hybridization

    Cell surface and cytoplasmic immunohistochemical staining of FG1 and FG3 transfectants. The parental cells and transfectants were stained with anti-FLAG MoAb and isotype control antibody. Bright-field represents the image of each specimen. CHO-β.FG1, CHO transfectant with hβ2m and FG1; CHO-β.FG3, CHO transfectant with hβ2m and FG3; SEC-β.FG1, SEC transfectant with hβ2m and FG1; SEC-β.FG3, SEC transfectant with hβ2m and FG3; 721.221-FG1, 721.221 transfectant with FG1; 721/221-FG3, 721/221 transfectant with FG3.
    Figure Legend Snippet: Cell surface and cytoplasmic immunohistochemical staining of FG1 and FG3 transfectants. The parental cells and transfectants were stained with anti-FLAG MoAb and isotype control antibody. Bright-field represents the image of each specimen. CHO-β.FG1, CHO transfectant with hβ2m and FG1; CHO-β.FG3, CHO transfectant with hβ2m and FG3; SEC-β.FG1, SEC transfectant with hβ2m and FG1; SEC-β.FG3, SEC transfectant with hβ2m and FG3; 721.221-FG1, 721.221 transfectant with FG1; 721/221-FG3, 721/221 transfectant with FG3.

    Techniques Used: Immunohistochemistry, Staining, Transfection, Size-exclusion Chromatography

    Related Articles

    Electrophoresis:

    Article Title: An RNA Interference Screen Identifies Druggable Regulators of MeCP2 Stability
    Article Snippet: .. Following electrophoresis, gels were transferred onto PVDF membranes and probed with rabbit anti-MeCP2 (1:3,000, Zoghbi lab, #0535) , rabbit anti-GST (1:2000, Sigma-Aldrich G7781), rabbit anti-HIPK2 pY361 (1:500, Invitrogen), rabbit anti-MeCP2 pS80 (1:500, Active Motif), rabbit anti-PPP2R1A (1:3,000, Abcam, ab154551), mouse anti-Tau (1:2500, Abcam ab80579), rabbit anti-Tau pS356 (1:1000, Abcam ab75603), mouse anti-GAPDH 6C5 (1:20,000, Advanced Immunochemicals, 2-RGM2), and mouse anti-Vinculin (1:10,000, Sigma-Aldrich). .. Secondary antibodies were mouse anti-rabbit horseradish peroxidase (HRP) (1:3,000, Jackson ImmunoResearch Labs, 211-032-171) and donkey anti-mouse HRP (1:50,000, Jackson ImmunoResearch Labs, 715-035-150).

    Western Blot:

    Article Title: Microtubule-Dependent Modulation of Adhesion Complex Composition
    Article Snippet: .. Antibodies Primary antibodies used in this study for Western blotting were specific for α5 -integrin (H-104 rabbit polyclonal antibody, Santa Cruz Biotechnology), αv -integrin (rabbit polyclonal antibody, Abcam), talin (8D4 mouse monoclonal antibody, Sigma-Aldrich), vinculin (hVIN-1 mouse monoclonal antibody, Sigma-Aldrich), paxillin (clone 349 mouse monoclonal antibody, BD Biosciences), ILK (EPR1592 rabbit polyclonal antibody, Abcam), PDLIM5 (rabbit polyclonal antibody, Abcam), filamin A (PM6/317 mouse monoclonal antibody, Abcam), ELKS (ELKS-30 mouse monoclonal antibody, Sigma-Aldrich), transferrin receptor (H68.4 mouse monoclonal antibody, Life Technologies) and BAK (rabbit polyclonal antibody, Sigma-Aldrich). .. Primary antibodies used in this study for immunofluorescence experiments were specific for α5 -integrin (mAb11 rat monoclonal antibody), tubulin (YL1/2 rat monoclonal antibody, Millipore), vinculin (FITC-conjugated hVIN-1 mouse monoclonal antibody, Sigma-Aldrich), αv β3 -integrin (LM609 mouse monoclonal antibody, Millipore), tensin-1 (H-300 rabbit polyclonal antibody, Santa Cruz Biotechnology), pTyr (P-Tyr-100 mouse monoclonal antibody) and fibronectin (F3648 rabbit polyclonal antibody, Sigma).

    Incubation:

    Article Title: A transcriptome map of cellular transformation by the fos oncogene
    Article Snippet: .. Fixed cells were incubated with primary mouse monoclonal anti-vinculin antibody (Sigma; dilution of 1:200) and phalloidin-congjugated to Alexa-568 (Molecular Probes). .. An anti-mouse secondary antibody (dilution of 1:1000) conjugated to Alexa-488 (Molecular Probes) was used to detect vinculin.

    Staining:

    Article Title: α-Catenin cytomechanics – role in cadherin-dependent adhesion and mechanotransduction
    Article Snippet: .. Immediately after bead twisting, cells were fixed with 4% w/v paraformaldehyde (PFA) for 15 min at room temperature, then permeabilized with 0.1% Triton X-100 for 5 min, blocked in 1% w/v BSA for 20 min, and stained with phalloidin, primary antibodies and secondary antibodies in 1% w/v BSA for 1 h. Primary antibodies included rabbit monoclonal anti-α-catenin antibody (Sigma), mouse monoclonal anti-vinculin antibody (Sigma), and mouse monoclonal anti-E-cadherin antibody (clone 36, BD Transduction Laboratories). .. Secondary antibodies were coupled to FITC (Sigma) or Alexa Fluor 594 (Invitrogen), and Rhodamine-phalloidin was from Invitrogen.

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    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: 99/100, based on 796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 antibody/product/Millipore
    Average 99 stars, based on 796 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag m2 antibody - by Bioz Stars, 2020-09
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    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

    Relative stability of NCPs containing H3 and H3.3. ( A ) NCPs were prepared from cells expressing either H3-Flag or H3.3-Flag. NCP monomers suspended in a solvent containing 150 mM NaCl were obtained by fractionation on a sucrose gradient (see Materials and Methods) in a solvent containing 80 mM NaCl and buffers. In each case, the sample was immunoprecipitated with antibody to Flag, histones were isolated and fractionated by gel electrophoresis, and Western blots of the samples were probed with antibody to H2A ( left ) or H2A.Z ( right ). Input lanes are loaded with an aliquot representing 10% of the starting sample. The label “150 mM/80 mM gradient” indicates the highest NaCl concentration used in NCP preparation and the NaCl concentration in the gradient. (Nab) No antibody control. ( B ) Comparative recoveries of H2A and H2A.Z from the data in A . The amounts of H2A or H2A.Z in H3-Flag-containing NCPs were set to 1. The relative amount was calculated by comparing the intensity of immunoprecipitated H2A or H2A.Z with that of immunoprecipitated H3-Flag or H3.3-Flag.

    Journal: Genes & Development

    Article Title: Nucleosome stability mediated by histone variants H3.3 and H2A.Z

    doi: 10.1101/gad.1547707

    Figure Lengend Snippet: Relative stability of NCPs containing H3 and H3.3. ( A ) NCPs were prepared from cells expressing either H3-Flag or H3.3-Flag. NCP monomers suspended in a solvent containing 150 mM NaCl were obtained by fractionation on a sucrose gradient (see Materials and Methods) in a solvent containing 80 mM NaCl and buffers. In each case, the sample was immunoprecipitated with antibody to Flag, histones were isolated and fractionated by gel electrophoresis, and Western blots of the samples were probed with antibody to H2A ( left ) or H2A.Z ( right ). Input lanes are loaded with an aliquot representing 10% of the starting sample. The label “150 mM/80 mM gradient” indicates the highest NaCl concentration used in NCP preparation and the NaCl concentration in the gradient. (Nab) No antibody control. ( B ) Comparative recoveries of H2A and H2A.Z from the data in A . The amounts of H2A or H2A.Z in H3-Flag-containing NCPs were set to 1. The relative amount was calculated by comparing the intensity of immunoprecipitated H2A or H2A.Z with that of immunoprecipitated H3-Flag or H3.3-Flag.

    Article Snippet: The antibodies used were as follows: anti-Flag M2 Monoclonal antibody (F3165), anti-histone H2A.Z (07-594), anti-histone H3 (05-499), anti-acetyl-histone H3 (06-599), anti-histone H2A (7-146), and anti-histone H2B (07-371).

    Techniques: Expressing, Fractionation, Immunoprecipitation, Isolation, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay

    ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z immunoprecipitation. Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.

    Journal: Genes & Development

    Article Title: Nucleosome stability mediated by histone variants H3.3 and H2A.Z

    doi: 10.1101/gad.1547707

    Figure Lengend Snippet: ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z immunoprecipitation. Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.

    Article Snippet: The antibodies used were as follows: anti-Flag M2 Monoclonal antibody (F3165), anti-histone H2A.Z (07-594), anti-histone H3 (05-499), anti-acetyl-histone H3 (06-599), anti-histone H2A (7-146), and anti-histone H2B (07-371).

    Techniques: Chromatin Immunoprecipitation, Expressing, Immunoprecipitation

    Progranulin Interacts with GCase. HEK-293 cells were co-transfected with constructs expressing HA-tagged human progranulin and/or myc-flag-tagged human GCase. HA-tagged progranulin was then immunoprecipitated from cell lysates with an anti-HA antibody. a , Flag-tagged GCase co-immunoprecipitated with progranulin, indicating interaction of the two proteins. b , Consistent with the co-immunoprecipitation of GCase with progranulin, we detected strong proximity ligation (PLA) signal in HEK-293 cells co-transfected with human progranulin and human GCase constructs. c , The specificity of the Flag-HA PLA signal was confirmed by the presence of significantly more PLA puncta from cells co-transfected with the progranulin-HA and GCase-Flag constructs than in cells transfected with only one of the constructs, or from cells that underwent PLA in the absence of the HA and Flag antibodies (ANOVA effect of experimental condition, p

    Journal: Acta Neuropathologica Communications

    Article Title: Impaired β-glucocerebrosidase activity and processing in frontotemporal dementia due to progranulin mutations

    doi: 10.1186/s40478-019-0872-6

    Figure Lengend Snippet: Progranulin Interacts with GCase. HEK-293 cells were co-transfected with constructs expressing HA-tagged human progranulin and/or myc-flag-tagged human GCase. HA-tagged progranulin was then immunoprecipitated from cell lysates with an anti-HA antibody. a , Flag-tagged GCase co-immunoprecipitated with progranulin, indicating interaction of the two proteins. b , Consistent with the co-immunoprecipitation of GCase with progranulin, we detected strong proximity ligation (PLA) signal in HEK-293 cells co-transfected with human progranulin and human GCase constructs. c , The specificity of the Flag-HA PLA signal was confirmed by the presence of significantly more PLA puncta from cells co-transfected with the progranulin-HA and GCase-Flag constructs than in cells transfected with only one of the constructs, or from cells that underwent PLA in the absence of the HA and Flag antibodies (ANOVA effect of experimental condition, p

    Article Snippet: Immunoprecipitates were blotted for Flag tag (mouse monoclonal, #F3165, MilliporeSigma) to detect co-immunoprecipitated GCase, and for progranulin to confirm successful pull down of HA-tagged progranulin (rabbit polyclonal, #40–3400, ThermoFisher).

    Techniques: Transfection, Construct, Expressing, Immunoprecipitation, Ligation, Proximity Ligation Assay

    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