• Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Name:
    FLIP Antibody
    Description:
    Cellular FLIP FLICE inhibitory protein is a regulator of apoptosis that has various names such as c FLIP 1 Casper 2 CLARP 3 FLAME 4 I FLICE 5 MRIT 6 CASH 7 and Usurpin 8 FLIP is expressed as two alternative splice isoforms FLIP short FLIPS and FLIP long FLIPL FLIPS contains two death effector domains DEDs like those found on the death receptor adaptor protein FADD and the pro domain of caspase 8 FLIPL shares significant homology with caspase 8 FLICE and contains an additional death effector domain but FLIPL lacks the catalytic active site of the caspases and does not have protease activity Both FLIP isoforms have been reported to interact with FADD and pro caspase 8 The role of FLIP in apoptosis is controversial as some research studies have reported it to be anti apoptotic while others claim that it is pro apoptotic Overexpression of FLIPL can lead to caspase 8 heterodimers that produce an active protease resulting in apoptosis However at physiological levels it is thought that the binding of FLIP to the DED of FADD results in inhibition of caspase 8 processing Reduction of FLIP by siRNA or gene targeting sensitizes cells to death receptor mediated apoptosis FLIP has also been implicated in the resistance of cancer cells to apoptosis and is upregulated in some cancer types including Hodgkin s lymphoma and ovarian and colon carcinomas 9
    Catalog Number:
    3210
    Price:
    None
    Applications:
    Western Blot
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding cysteine 155 of FLIP. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti flip
    <t>FLIP</t> activates the canonical NF-κB pathway in myeloid cells. a Suppressive activity of THP1 cells transfected with c-FLIP RNA was measured by enumeration of an absolute number of human CD3 + T cells collected after in vitro co-culture. b Immunofluorescence confocal microscopy of <t>p65</t> (red), p50 and p52 (green) nuclear translocation in transfected THP1 cells. c Nuclear c-FLIP protein expression by western blot in THP1 cells transfected with either GFP or c-FLIP RNA at different time points. d Immunofluorescence confocal microscopy of p50 (red) and c-FLIP (green) nuclear translocation in transfected THP1 cells. e CD11b + Ly6C + cells (M-MDSCs) were isolated from the spleen of MCA203 tumor-bearing, wild-type mice by flow sorter and transfected for 18 h with scramble, IKKα, IKKβ, or the combination IKKα plus IKKβ siRNAs. After transfection, cells were washed three times. M-MDSCs were co-incubated with peptide-stimulated CellTrace-labeled OT-I cells. Suppressive activity was measured by enumerating absolute numbers of CD8 + T cells collected after in vitro co-culture. f PD-L1 expression in transfected M-MDSCs. Data are presented either as mean ± s.e.m of three independent experiments ( a ) or as mean ± s.e.m of four independent experimental transfections ( e , f ) where each plot refers to CD11b + Ly6C + cells isolated from the pooled spleens of three tumor-bearing mice. Original images × 800 for all panels ( b , d ). * P
    Cellular FLIP FLICE inhibitory protein is a regulator of apoptosis that has various names such as c FLIP 1 Casper 2 CLARP 3 FLAME 4 I FLICE 5 MRIT 6 CASH 7 and Usurpin 8 FLIP is expressed as two alternative splice isoforms FLIP short FLIPS and FLIP long FLIPL FLIPS contains two death effector domains DEDs like those found on the death receptor adaptor protein FADD and the pro domain of caspase 8 FLIPL shares significant homology with caspase 8 FLICE and contains an additional death effector domain but FLIPL lacks the catalytic active site of the caspases and does not have protease activity Both FLIP isoforms have been reported to interact with FADD and pro caspase 8 The role of FLIP in apoptosis is controversial as some research studies have reported it to be anti apoptotic while others claim that it is pro apoptotic Overexpression of FLIPL can lead to caspase 8 heterodimers that produce an active protease resulting in apoptosis However at physiological levels it is thought that the binding of FLIP to the DED of FADD results in inhibition of caspase 8 processing Reduction of FLIP by siRNA or gene targeting sensitizes cells to death receptor mediated apoptosis FLIP has also been implicated in the resistance of cancer cells to apoptosis and is upregulated in some cancer types including Hodgkin s lymphoma and ovarian and colon carcinomas 9
    https://www.bioz.com/result/anti flip/product/Cell Signaling Technology Inc
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    anti flip - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "Induction of immunosuppressive functions and NF-κB by FLIP in monocytes"

    Article Title: Induction of immunosuppressive functions and NF-κB by FLIP in monocytes

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07654-4

    FLIP activates the canonical NF-κB pathway in myeloid cells. a Suppressive activity of THP1 cells transfected with c-FLIP RNA was measured by enumeration of an absolute number of human CD3 + T cells collected after in vitro co-culture. b Immunofluorescence confocal microscopy of p65 (red), p50 and p52 (green) nuclear translocation in transfected THP1 cells. c Nuclear c-FLIP protein expression by western blot in THP1 cells transfected with either GFP or c-FLIP RNA at different time points. d Immunofluorescence confocal microscopy of p50 (red) and c-FLIP (green) nuclear translocation in transfected THP1 cells. e CD11b + Ly6C + cells (M-MDSCs) were isolated from the spleen of MCA203 tumor-bearing, wild-type mice by flow sorter and transfected for 18 h with scramble, IKKα, IKKβ, or the combination IKKα plus IKKβ siRNAs. After transfection, cells were washed three times. M-MDSCs were co-incubated with peptide-stimulated CellTrace-labeled OT-I cells. Suppressive activity was measured by enumerating absolute numbers of CD8 + T cells collected after in vitro co-culture. f PD-L1 expression in transfected M-MDSCs. Data are presented either as mean ± s.e.m of three independent experiments ( a ) or as mean ± s.e.m of four independent experimental transfections ( e , f ) where each plot refers to CD11b + Ly6C + cells isolated from the pooled spleens of three tumor-bearing mice. Original images × 800 for all panels ( b , d ). * P
    Figure Legend Snippet: FLIP activates the canonical NF-κB pathway in myeloid cells. a Suppressive activity of THP1 cells transfected with c-FLIP RNA was measured by enumeration of an absolute number of human CD3 + T cells collected after in vitro co-culture. b Immunofluorescence confocal microscopy of p65 (red), p50 and p52 (green) nuclear translocation in transfected THP1 cells. c Nuclear c-FLIP protein expression by western blot in THP1 cells transfected with either GFP or c-FLIP RNA at different time points. d Immunofluorescence confocal microscopy of p50 (red) and c-FLIP (green) nuclear translocation in transfected THP1 cells. e CD11b + Ly6C + cells (M-MDSCs) were isolated from the spleen of MCA203 tumor-bearing, wild-type mice by flow sorter and transfected for 18 h with scramble, IKKα, IKKβ, or the combination IKKα plus IKKβ siRNAs. After transfection, cells were washed three times. M-MDSCs were co-incubated with peptide-stimulated CellTrace-labeled OT-I cells. Suppressive activity was measured by enumerating absolute numbers of CD8 + T cells collected after in vitro co-culture. f PD-L1 expression in transfected M-MDSCs. Data are presented either as mean ± s.e.m of three independent experiments ( a ) or as mean ± s.e.m of four independent experimental transfections ( e , f ) where each plot refers to CD11b + Ly6C + cells isolated from the pooled spleens of three tumor-bearing mice. Original images × 800 for all panels ( b , d ). * P

    Techniques Used: Activity Assay, Transfection, In Vitro, Co-Culture Assay, Immunofluorescence, Confocal Microscopy, Translocation Assay, Expressing, Western Blot, Isolation, Mouse Assay, Flow Cytometry, Incubation, Labeling

    2) Product Images from "Cell fate regulation by gelsolin in human gynecologic cancers"

    Article Title: Cell fate regulation by gelsolin in human gynecologic cancers

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

    doi: 10.1073/pnas.1401166111

    A hypothetical model illustrating the role and regulation of GSN in the control of chemosensitivity in cancer cells. In nonstress state, GSN forms a complex with FLIP and Itch. CDDP leads to the dissociation of GSN from the GSN-FLIP-Itch complex in sensitive
    Figure Legend Snippet: A hypothetical model illustrating the role and regulation of GSN in the control of chemosensitivity in cancer cells. In nonstress state, GSN forms a complex with FLIP and Itch. CDDP leads to the dissociation of GSN from the GSN-FLIP-Itch complex in sensitive

    Techniques Used:

    CDDP attenuates FLIP-GSN colocalization at OV2008 but not C13* cells. Confocal imaging of double-stained FLIP and GSN in OV2008 and C13* cells. FLIP ( A1 ) distributed throughout the cytoplasm and nucleus in OV2008 treated with DMSO (control). CDDP treatment
    Figure Legend Snippet: CDDP attenuates FLIP-GSN colocalization at OV2008 but not C13* cells. Confocal imaging of double-stained FLIP and GSN in OV2008 and C13* cells. FLIP ( A1 ) distributed throughout the cytoplasm and nucleus in OV2008 treated with DMSO (control). CDDP treatment

    Techniques Used: Imaging, Staining

    CDDP attenuates the GSN-FLIP-Itch interaction in chemosensitive but not resistant counterparts. The FLIP-GSN-Itch interaction was attenuated by CDDP in chemosensitive (OV2008) but not resistant (C13*) cells as detected by immunocoprecipitation. Cells
    Figure Legend Snippet: CDDP attenuates the GSN-FLIP-Itch interaction in chemosensitive but not resistant counterparts. The FLIP-GSN-Itch interaction was attenuated by CDDP in chemosensitive (OV2008) but not resistant (C13*) cells as detected by immunocoprecipitation. Cells

    Techniques Used:

    3) Product Images from "Induction of immunosuppressive functions and NF-κB by FLIP in monocytes"

    Article Title: Induction of immunosuppressive functions and NF-κB by FLIP in monocytes

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07654-4

    FLIP activates the canonical NF-κB pathway in myeloid cells. a Suppressive activity of THP1 cells transfected with c-FLIP RNA was measured by enumeration of an absolute number of human CD3 + T cells collected after in vitro co-culture. b Immunofluorescence confocal microscopy of p65 (red), p50 and p52 (green) nuclear translocation in transfected THP1 cells. c Nuclear c-FLIP protein expression by western blot in THP1 cells transfected with either GFP or c-FLIP RNA at different time points. d Immunofluorescence confocal microscopy of p50 (red) and c-FLIP (green) nuclear translocation in transfected THP1 cells. e CD11b + Ly6C + cells (M-MDSCs) were isolated from the spleen of MCA203 tumor-bearing, wild-type mice by flow sorter and transfected for 18 h with scramble, IKKα, IKKβ, or the combination IKKα plus IKKβ siRNAs. After transfection, cells were washed three times. M-MDSCs were co-incubated with peptide-stimulated CellTrace-labeled OT-I cells. Suppressive activity was measured by enumerating absolute numbers of CD8 + T cells collected after in vitro co-culture. f PD-L1 expression in transfected M-MDSCs. Data are presented either as mean ± s.e.m of three independent experiments ( a ) or as mean ± s.e.m of four independent experimental transfections ( e , f ) where each plot refers to CD11b + Ly6C + cells isolated from the pooled spleens of three tumor-bearing mice. Original images × 800 for all panels ( b , d ). * P
    Figure Legend Snippet: FLIP activates the canonical NF-κB pathway in myeloid cells. a Suppressive activity of THP1 cells transfected with c-FLIP RNA was measured by enumeration of an absolute number of human CD3 + T cells collected after in vitro co-culture. b Immunofluorescence confocal microscopy of p65 (red), p50 and p52 (green) nuclear translocation in transfected THP1 cells. c Nuclear c-FLIP protein expression by western blot in THP1 cells transfected with either GFP or c-FLIP RNA at different time points. d Immunofluorescence confocal microscopy of p50 (red) and c-FLIP (green) nuclear translocation in transfected THP1 cells. e CD11b + Ly6C + cells (M-MDSCs) were isolated from the spleen of MCA203 tumor-bearing, wild-type mice by flow sorter and transfected for 18 h with scramble, IKKα, IKKβ, or the combination IKKα plus IKKβ siRNAs. After transfection, cells were washed three times. M-MDSCs were co-incubated with peptide-stimulated CellTrace-labeled OT-I cells. Suppressive activity was measured by enumerating absolute numbers of CD8 + T cells collected after in vitro co-culture. f PD-L1 expression in transfected M-MDSCs. Data are presented either as mean ± s.e.m of three independent experiments ( a ) or as mean ± s.e.m of four independent experimental transfections ( e , f ) where each plot refers to CD11b + Ly6C + cells isolated from the pooled spleens of three tumor-bearing mice. Original images × 800 for all panels ( b , d ). * P

    Techniques Used: Activity Assay, Transfection, In Vitro, Co-Culture Assay, Immunofluorescence, Confocal Microscopy, Translocation Assay, Expressing, Western Blot, Isolation, Mouse Assay, Flow Cytometry, Incubation, Labeling

    4) Product Images from "Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network"

    Article Title: Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007305

    c-FLIP is an essential mediator of basal apoptosis in human cancer cells and functions downstream of GSK3, Fbw7, c-Jun and JNK1. (A) Levels of apoptosis observed following co-silencing of c-FLIP with pro-apoptotic mediators. (B) Protein levels showing pro-caspase 8 cleavage and c-Jun accumulation following c-FLIP depletion by RNAi.
    Figure Legend Snippet: c-FLIP is an essential mediator of basal apoptosis in human cancer cells and functions downstream of GSK3, Fbw7, c-Jun and JNK1. (A) Levels of apoptosis observed following co-silencing of c-FLIP with pro-apoptotic mediators. (B) Protein levels showing pro-caspase 8 cleavage and c-Jun accumulation following c-FLIP depletion by RNAi.

    Techniques Used:

    5) Product Images from "Catalytic activity of the caspase-8-FLIPL complex inhibits RIPK3-dependent necrosis"

    Article Title: Catalytic activity of the caspase-8-FLIPL complex inhibits RIPK3-dependent necrosis

    Journal: Nature

    doi: 10.1038/nature09852

    Both catalytically active caspase-8 and FLIP are required for suppression of TNF-induced RIPK3-dependent death a. Fluorogenic substrate cleavage activity of recombinant purified FKBP-caspase-8 WT or non-cleavable FKBP-caspase-8 DA in the presence of recombinant purified FRB-FLIP, a compound that induces FKBP-FRB heterodimers (heterodimerizer), or FRB-FLIP and heterodimerizer. ND indicates none detected. (Error bars are s.d., n=3) b. Western blot analysis of RIPK3 −/− :Caspase-8 −/− (DKO) MEF stably expressing the indicated mutants of caspase-8 and RIPK3. Caspase-8 CA indicates catalytically inactive caspase-8. c, d. Cell death assessed by propidium iodide (PI) uptake of DKO MEF expressing the indicated caspase-8 mutants in the absence ( c ) or presence ( d ) of stably expressed RIPK3, following transfection with scramble or FLIP-targeted siRNA, and 12 hours TNF treatment. (Error bars are s.d., n=3) e, f. Cell death (PI uptake) of SVEC 4–10 cells stably expressing RIPK3 specific ( e ) or scramble ( f ) shRNA, transfected with the indicated siRNAs and treated with TNF for 12 hours. (Graph is mean of 2 independent experiments, error bars indicate range) g. Cell death (PI uptake) of L929 cells expressing scramble or RIPK3-specific shRNA, transfected with siRNAs specific for caspase-8 and/or FLIP as indicated. Death was assessed 48h post-transfection. (Error bars are s.d., n=3.) The data presented are representative of results obtained with either of 2 separate siRNAs to both caspase-8 and FLIP.
    Figure Legend Snippet: Both catalytically active caspase-8 and FLIP are required for suppression of TNF-induced RIPK3-dependent death a. Fluorogenic substrate cleavage activity of recombinant purified FKBP-caspase-8 WT or non-cleavable FKBP-caspase-8 DA in the presence of recombinant purified FRB-FLIP, a compound that induces FKBP-FRB heterodimers (heterodimerizer), or FRB-FLIP and heterodimerizer. ND indicates none detected. (Error bars are s.d., n=3) b. Western blot analysis of RIPK3 −/− :Caspase-8 −/− (DKO) MEF stably expressing the indicated mutants of caspase-8 and RIPK3. Caspase-8 CA indicates catalytically inactive caspase-8. c, d. Cell death assessed by propidium iodide (PI) uptake of DKO MEF expressing the indicated caspase-8 mutants in the absence ( c ) or presence ( d ) of stably expressed RIPK3, following transfection with scramble or FLIP-targeted siRNA, and 12 hours TNF treatment. (Error bars are s.d., n=3) e, f. Cell death (PI uptake) of SVEC 4–10 cells stably expressing RIPK3 specific ( e ) or scramble ( f ) shRNA, transfected with the indicated siRNAs and treated with TNF for 12 hours. (Graph is mean of 2 independent experiments, error bars indicate range) g. Cell death (PI uptake) of L929 cells expressing scramble or RIPK3-specific shRNA, transfected with siRNAs specific for caspase-8 and/or FLIP as indicated. Death was assessed 48h post-transfection. (Error bars are s.d., n=3.) The data presented are representative of results obtained with either of 2 separate siRNAs to both caspase-8 and FLIP.

    Techniques Used: Activity Assay, Recombinant, Purification, Western Blot, Stable Transfection, Expressing, Transfection, shRNA

    The FLIP-Caspase-8 heterodimeric complex suppresses RIPK3-dependent cell death a . Inhibitory effect of CrmA on FKBP-Caspase-8 homodimers or caspase-8 DA -FLIP heterodimers induced by dimerizer treatment of purified recombinant protein. b . Cell death (PI uptake) of RIPK3-deficient (NIH) or RIPK3 expressing (SA) 3T3 cells treated as indicated for 12h. (Error bars are s.d., n=3) c, d, e. Cell death (PI uptake) of 3T3-SA cells stably expressing vector ( c ) or CrmA ( d ), or anti-apoptotic Bcl-XL ( e ) following transfection with the indicated siRNA and treatment with TNF and the RIPK1 inhibitor Nec-1 as indicated for 24h. (Error bars are s.d., n=3) f. 3T3-SA cells expressing Bcl-XL were subjected to immunoprecipitation of FADD following transfection of siRNAs to caspase-8 and FLIP and treatment with TNF for 90 minutes as indicated. Immune complexes were resolved by western blotting with the indicated antibodies. The data presented are representative of results obtained with either of 2 separate siRNAs to both caspase-8 and FLIP.
    Figure Legend Snippet: The FLIP-Caspase-8 heterodimeric complex suppresses RIPK3-dependent cell death a . Inhibitory effect of CrmA on FKBP-Caspase-8 homodimers or caspase-8 DA -FLIP heterodimers induced by dimerizer treatment of purified recombinant protein. b . Cell death (PI uptake) of RIPK3-deficient (NIH) or RIPK3 expressing (SA) 3T3 cells treated as indicated for 12h. (Error bars are s.d., n=3) c, d, e. Cell death (PI uptake) of 3T3-SA cells stably expressing vector ( c ) or CrmA ( d ), or anti-apoptotic Bcl-XL ( e ) following transfection with the indicated siRNA and treatment with TNF and the RIPK1 inhibitor Nec-1 as indicated for 24h. (Error bars are s.d., n=3) f. 3T3-SA cells expressing Bcl-XL were subjected to immunoprecipitation of FADD following transfection of siRNAs to caspase-8 and FLIP and treatment with TNF for 90 minutes as indicated. Immune complexes were resolved by western blotting with the indicated antibodies. The data presented are representative of results obtained with either of 2 separate siRNAs to both caspase-8 and FLIP.

    Techniques Used: Purification, Recombinant, Expressing, Stable Transfection, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot

    6) Product Images from "TAK1 regulates caspase 8 activation and necroptotic signaling via multiple cell death checkpoints"

    Article Title: TAK1 regulates caspase 8 activation and necroptotic signaling via multiple cell death checkpoints

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.294

    Inhibition of TAK1 triggers two caspase 8 activation pathways through induction of the RIP1-FADD-caspase 8 complex as well as FLIP degradation. ( a ) Caspase 8 activity in wild-type MEFs treated as indicated for 4 h. † P
    Figure Legend Snippet: Inhibition of TAK1 triggers two caspase 8 activation pathways through induction of the RIP1-FADD-caspase 8 complex as well as FLIP degradation. ( a ) Caspase 8 activity in wild-type MEFs treated as indicated for 4 h. † P

    Techniques Used: Inhibition, Activation Assay, Activity Assay

    Related Articles

    Western Blot:

    Article Title: Catalytic activity of the caspase-8-FLIPL complex inhibits RIPK3-dependent necrosis
    Article Snippet: .. Compounds, antibodies and cytokines Antibodies used for western blot were: Anti-RIPK1 from BD (610458), anti-RIPK3 from Imgenex (IMG-5523-2), anti-Caspase-8 (1G12) and anti-FLIP (Dave-2) both from Alexis, and anti-Actin (C4) from MP, anti-Caspase-3 from Cell Signaling (9662), and anti-Bcl-XL(S18) and anti-FADD(M19) both from Santa Cruz. .. Murine TNF-α came from Peprotech.

    Incubation:

    Article Title: Downregulation of TACC3 inhibits tumor growth and migration in osteosarcoma cells through regulation of the NF-κB signaling pathway
    Article Snippet: .. After blocking non-specific binding sites for 60 min with 8% non-fat milk in TBST, the membranes were incubated with primary antibodies, rabbit polyclonal anti-TACC3 (1:1,000; Abcam, Cambridge, UK; cat. no. ab134154), anti-p65 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 8242) anti-matrix metalloproteinase-9 (MMP9; 1:1,000; Cell Signaling Technology, Inc.; cat. no. 13667), anti-cellular FLICE-like inhibitory protein (c-FLIP; 1:500; Cell Signaling Technology, Inc.; cat. no. 56343) or GAPDH (1:10,000; ProteinTech Group, Inc., Chicago, IL, USA; cat. no. 10494-1-AP) overnight at 4°C. .. The membranes were washed four times with TRIS-buffered saline with Tween-20 for 10 min. After washing, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000; EMD/Merck KGaA, Darmstadt, Germany; cat. no. AP307P) at room temperature for 1 h, and an enhanced chemiluminescence detection kit (Cell Signaling Technology, Inc.), was used to visualize the proteins.

    Article Title: Cell fate regulation by gelsolin in human gynecologic cancers
    Article Snippet: .. OV2008 cells were fixed and then incubated with anti-GSN (Sigma-Aldrich) and anti-FLIP (Cell Signaling) and subsequently with secondary donkey-conjugated antibodies: anti-goat (Cy5), anti-mouse (Cy3), and anti-rabbit (FITC), as previously reported ( ). ..

    Article Title: Induction of immunosuppressive functions and NF-κB by FLIP in monocytes
    Article Snippet: .. We used the following antibodies: anti-mouse caspase-3 (D3E9), caspase-7 (D6H1), caspase-8 (D35G2) and caspase-9 (D2D4), anti-human p65 (D14E12), p50 (D7H5M), p52 (18D10), H3 (D1H2), anti-FLIP (D5J1E) purchased from Cell Signaling Technologies (Danvers, MA, USA), followed by incubation with the secondary goat anti-rabbit IgG antibody, horseradish peroxidase (HRP)-conjugated (Millipore, Billerica, MA, USA). .. Uncropped images of immunoblots displayed in the figures are in the Supplemntary section (Supplementary Fig. ).

    Binding Assay:

    Article Title: Downregulation of TACC3 inhibits tumor growth and migration in osteosarcoma cells through regulation of the NF-κB signaling pathway
    Article Snippet: .. After blocking non-specific binding sites for 60 min with 8% non-fat milk in TBST, the membranes were incubated with primary antibodies, rabbit polyclonal anti-TACC3 (1:1,000; Abcam, Cambridge, UK; cat. no. ab134154), anti-p65 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 8242) anti-matrix metalloproteinase-9 (MMP9; 1:1,000; Cell Signaling Technology, Inc.; cat. no. 13667), anti-cellular FLICE-like inhibitory protein (c-FLIP; 1:500; Cell Signaling Technology, Inc.; cat. no. 56343) or GAPDH (1:10,000; ProteinTech Group, Inc., Chicago, IL, USA; cat. no. 10494-1-AP) overnight at 4°C. .. The membranes were washed four times with TRIS-buffered saline with Tween-20 for 10 min. After washing, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000; EMD/Merck KGaA, Darmstadt, Germany; cat. no. AP307P) at room temperature for 1 h, and an enhanced chemiluminescence detection kit (Cell Signaling Technology, Inc.), was used to visualize the proteins.

    Blocking Assay:

    Article Title: Downregulation of TACC3 inhibits tumor growth and migration in osteosarcoma cells through regulation of the NF-κB signaling pathway
    Article Snippet: .. After blocking non-specific binding sites for 60 min with 8% non-fat milk in TBST, the membranes were incubated with primary antibodies, rabbit polyclonal anti-TACC3 (1:1,000; Abcam, Cambridge, UK; cat. no. ab134154), anti-p65 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 8242) anti-matrix metalloproteinase-9 (MMP9; 1:1,000; Cell Signaling Technology, Inc.; cat. no. 13667), anti-cellular FLICE-like inhibitory protein (c-FLIP; 1:500; Cell Signaling Technology, Inc.; cat. no. 56343) or GAPDH (1:10,000; ProteinTech Group, Inc., Chicago, IL, USA; cat. no. 10494-1-AP) overnight at 4°C. .. The membranes were washed four times with TRIS-buffered saline with Tween-20 for 10 min. After washing, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000; EMD/Merck KGaA, Darmstadt, Germany; cat. no. AP307P) at room temperature for 1 h, and an enhanced chemiluminescence detection kit (Cell Signaling Technology, Inc.), was used to visualize the proteins.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Cell Signaling Technology Inc mouse anti p21
    SIRT6 regulates TGF-β-induced cellular senescence via proteasomal degradation of <t>p21</t> in HBEC. SIRT6 suppresses p21 expression. A : RT-PCR using primers to p21 and β-actin was performed from control empty vector transfected ( lane 1 ), control empty vector transfected in the presense of TGF-β ( lane 2 ), and SIRT6 expression vector transfected in the presense of TGF-β ( lane 3 ). B , top : WB using anti-p21 in control empty vector and nonsilencing control siRNA transfected ( lanes 1 and 4 ), SIRT6 expression vector and nonsilencing control siRNA transfected ( lanes 2 and 5 ), and SIRT6 siRNA and control empty vector transfected ( lanes 3 and 6 ) HBEC, in the absence ( lanes 1 , 2 , and 3 ) or in the presence ( lanes 4 , 5 , and 6 ) of TGF-β (2 ng/ml). Bottom : average (±SE) taken from 5 independent WB shown as relative expression of p21 compared with β-actin. Open bar is control empty vector and nonsilencing control siRNA transfected, solid bar is SIRT6 expression vector and nonsilencing control siRNA transfected, and striped bar is SIRT6 siRNA and control empty vector transfected. * P
    Mouse Anti P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti p21/product/Cell Signaling Technology Inc
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    mouse anti p21 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc mouse iso igg antibody conjugated sepharose beads
    CaM was recruited into TRA-8 activated DISC in triple-negative MDA-MB-231 and ER-positive ZR-75-1 breast cancer cells Co-immunoprecipitation of DR5, CaM, FADD and Caspase-8 from MDA-MB-231 (A) and ZR-75-1 cells (B) treated with 1 μg/mL TRA-8 3 hours. Cell Lysate lane: whole cell lysate of breast cancer cells before IP. IP: DISC lane: Co-IP of CaM with DR5-mediated DISC from cell lysates using anti-DR5 (D-6) antibody with protein A/G Plus-Agarose beads, mouse <t>iso-IgG</t> conjugated <t>sepharose</t> beads, or 4B-sepharose beads. IP D-6 lane: IP using anti-DR5 (D-6) antibody with protein A/G Plus-Agarose beads, IP IsoIgG lane: IP using a mouse Iso-IgG conjugated sepharose beads. IP S lane: IP result using 4B-sepharose beads. Representative results are from two independent experiments.
    Mouse Iso Igg Antibody Conjugated Sepharose Beads, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse iso igg antibody conjugated sepharose beads/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse iso igg antibody conjugated sepharose beads - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc alexa fluor 488 conjugated anti mouse igg
    Epitope mapping of LpMab-7 by Western blot analysis and flow cytometry. ( A ) Western blotting by LpMab-3, LpMab-7, NZ-1, r2336, 1E6, RMab-3 (α-IDH1), and AC-15 (α-β-actin). Total cell lysate were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/mL of primary antibodies and then with peroxidase-conjugated secondary antibodies; the membrane was detected using a Sayaca-Imager. Blue arrow, 40 kDa band (glycosylated); red arrow, 30 kDa band (glycosylated); black arrow, 25 kDa band (non-glycosylated). ( B ) Point mutants of human podoplanin were treated with NZ-1 and LpMab-3 (1 μg/mL) for 30 min at 4°C, followed by treatment with <t>Alexa</t> <t>Fluor</t> 488 conjugated anti-rat <t>IgG</t> and anti-mouse IgG, respectively. Fluorescence data were collected using a Cell Analyzer EC800. ( C ) TGIRIE sequence and α2–6 linked sialic acid are the critical epitope of LpMab-3.
    Alexa Fluor 488 Conjugated Anti Mouse Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488 conjugated anti mouse igg/product/Cell Signaling Technology Inc
    Average 99 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 488 conjugated anti mouse igg - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    SIRT6 regulates TGF-β-induced cellular senescence via proteasomal degradation of p21 in HBEC. SIRT6 suppresses p21 expression. A : RT-PCR using primers to p21 and β-actin was performed from control empty vector transfected ( lane 1 ), control empty vector transfected in the presense of TGF-β ( lane 2 ), and SIRT6 expression vector transfected in the presense of TGF-β ( lane 3 ). B , top : WB using anti-p21 in control empty vector and nonsilencing control siRNA transfected ( lanes 1 and 4 ), SIRT6 expression vector and nonsilencing control siRNA transfected ( lanes 2 and 5 ), and SIRT6 siRNA and control empty vector transfected ( lanes 3 and 6 ) HBEC, in the absence ( lanes 1 , 2 , and 3 ) or in the presence ( lanes 4 , 5 , and 6 ) of TGF-β (2 ng/ml). Bottom : average (±SE) taken from 5 independent WB shown as relative expression of p21 compared with β-actin. Open bar is control empty vector and nonsilencing control siRNA transfected, solid bar is SIRT6 expression vector and nonsilencing control siRNA transfected, and striped bar is SIRT6 siRNA and control empty vector transfected. * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-?-induced senescence of human bronchial epithelial cells

    doi: 10.1152/ajplung.00097.2010

    Figure Lengend Snippet: SIRT6 regulates TGF-β-induced cellular senescence via proteasomal degradation of p21 in HBEC. SIRT6 suppresses p21 expression. A : RT-PCR using primers to p21 and β-actin was performed from control empty vector transfected ( lane 1 ), control empty vector transfected in the presense of TGF-β ( lane 2 ), and SIRT6 expression vector transfected in the presense of TGF-β ( lane 3 ). B , top : WB using anti-p21 in control empty vector and nonsilencing control siRNA transfected ( lanes 1 and 4 ), SIRT6 expression vector and nonsilencing control siRNA transfected ( lanes 2 and 5 ), and SIRT6 siRNA and control empty vector transfected ( lanes 3 and 6 ) HBEC, in the absence ( lanes 1 , 2 , and 3 ) or in the presence ( lanes 4 , 5 , and 6 ) of TGF-β (2 ng/ml). Bottom : average (±SE) taken from 5 independent WB shown as relative expression of p21 compared with β-actin. Open bar is control empty vector and nonsilencing control siRNA transfected, solid bar is SIRT6 expression vector and nonsilencing control siRNA transfected, and striped bar is SIRT6 siRNA and control empty vector transfected. * P

    Article Snippet: Antibodies used were rabbit anti-SIRT6 (Cell Signaling Technology, Beverly, MA), mouse anti-p21 (Cell Signaling Technology), rabbit anti-p53 (Cell Signaling Technology), goat anti-type I collagen (Southern Biotech), and alexa fluor 546 anti-rabbit IgG (Invitrogen).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, Western Blot

    p21 expression in IPF lung tissues. Immunohistochemical staining of p21 in IPF lung tissue is shown. Photomicrographs are of normal ( A ), mild-to-moderate fibrosis ( B ), FF ( C and D ), and dense fibrosis areas ( E ). F : percentage (±SE) of p21-positive epithelial cells in total epithelial cells from five cases. ** P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-?-induced senescence of human bronchial epithelial cells

    doi: 10.1152/ajplung.00097.2010

    Figure Lengend Snippet: p21 expression in IPF lung tissues. Immunohistochemical staining of p21 in IPF lung tissue is shown. Photomicrographs are of normal ( A ), mild-to-moderate fibrosis ( B ), FF ( C and D ), and dense fibrosis areas ( E ). F : percentage (±SE) of p21-positive epithelial cells in total epithelial cells from five cases. ** P

    Article Snippet: Antibodies used were rabbit anti-SIRT6 (Cell Signaling Technology, Beverly, MA), mouse anti-p21 (Cell Signaling Technology), rabbit anti-p53 (Cell Signaling Technology), goat anti-type I collagen (Southern Biotech), and alexa fluor 546 anti-rabbit IgG (Invitrogen).

    Techniques: Expressing, Immunohistochemistry, Staining

    Transforming growth factor (TGF)-β induces cellular senescence via p21 expression in human bronchial epithelial cells (HBEC). A , top : photograph of SA-β-gal staining of control ( left ) or TGF-β-treated (2 ng/ml for 48 h; right ) HBECs (original magnification, ×100). Bottom : percentage (±SE) of SA-β-gal-positive cells from 3 independent experiments. Open bar is no treatment, solid bar is TGF-β (2 ng/ml for 48 h), and striped bar is TGF-β (6 ng/ml for 48 h). * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-?-induced senescence of human bronchial epithelial cells

    doi: 10.1152/ajplung.00097.2010

    Figure Lengend Snippet: Transforming growth factor (TGF)-β induces cellular senescence via p21 expression in human bronchial epithelial cells (HBEC). A , top : photograph of SA-β-gal staining of control ( left ) or TGF-β-treated (2 ng/ml for 48 h; right ) HBECs (original magnification, ×100). Bottom : percentage (±SE) of SA-β-gal-positive cells from 3 independent experiments. Open bar is no treatment, solid bar is TGF-β (2 ng/ml for 48 h), and striped bar is TGF-β (6 ng/ml for 48 h). * P

    Article Snippet: Antibodies used were rabbit anti-SIRT6 (Cell Signaling Technology, Beverly, MA), mouse anti-p21 (Cell Signaling Technology), rabbit anti-p53 (Cell Signaling Technology), goat anti-type I collagen (Southern Biotech), and alexa fluor 546 anti-rabbit IgG (Invitrogen).

    Techniques: Expressing, Staining

    CaM was recruited into TRA-8 activated DISC in triple-negative MDA-MB-231 and ER-positive ZR-75-1 breast cancer cells Co-immunoprecipitation of DR5, CaM, FADD and Caspase-8 from MDA-MB-231 (A) and ZR-75-1 cells (B) treated with 1 μg/mL TRA-8 3 hours. Cell Lysate lane: whole cell lysate of breast cancer cells before IP. IP: DISC lane: Co-IP of CaM with DR5-mediated DISC from cell lysates using anti-DR5 (D-6) antibody with protein A/G Plus-Agarose beads, mouse iso-IgG conjugated sepharose beads, or 4B-sepharose beads. IP D-6 lane: IP using anti-DR5 (D-6) antibody with protein A/G Plus-Agarose beads, IP IsoIgG lane: IP using a mouse Iso-IgG conjugated sepharose beads. IP S lane: IP result using 4B-sepharose beads. Representative results are from two independent experiments.

    Journal: Journal of cellular biochemistry

    Article Title: Calmodulin Binding to Death Receptor 5-mediated Death-inducing Signaling Complex in Breast Cancer Cells

    doi: 10.1002/jcb.25882

    Figure Lengend Snippet: CaM was recruited into TRA-8 activated DISC in triple-negative MDA-MB-231 and ER-positive ZR-75-1 breast cancer cells Co-immunoprecipitation of DR5, CaM, FADD and Caspase-8 from MDA-MB-231 (A) and ZR-75-1 cells (B) treated with 1 μg/mL TRA-8 3 hours. Cell Lysate lane: whole cell lysate of breast cancer cells before IP. IP: DISC lane: Co-IP of CaM with DR5-mediated DISC from cell lysates using anti-DR5 (D-6) antibody with protein A/G Plus-Agarose beads, mouse iso-IgG conjugated sepharose beads, or 4B-sepharose beads. IP D-6 lane: IP using anti-DR5 (D-6) antibody with protein A/G Plus-Agarose beads, IP IsoIgG lane: IP using a mouse Iso-IgG conjugated sepharose beads. IP S lane: IP result using 4B-sepharose beads. Representative results are from two independent experiments.

    Article Snippet: As control experiments, MDA-MB-231 or ZR-75-1 cell lysates were incubated with mouse iso-IgG antibody conjugated sepharose beads (Cell Signaling Technology, Beverly, MA, specificity: none, source: mouse, Cat. 3420S, Lot# 5) or only 4B-sepharose beads (Sigma-Aldrich, St. Louis MO) overnight at 4 °C.

    Techniques: Chick Chorioallantoic Membrane Assay, Multiple Displacement Amplification, Immunoprecipitation, Co-Immunoprecipitation Assay

    Epitope mapping of LpMab-7 by Western blot analysis and flow cytometry. ( A ) Western blotting by LpMab-3, LpMab-7, NZ-1, r2336, 1E6, RMab-3 (α-IDH1), and AC-15 (α-β-actin). Total cell lysate were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/mL of primary antibodies and then with peroxidase-conjugated secondary antibodies; the membrane was detected using a Sayaca-Imager. Blue arrow, 40 kDa band (glycosylated); red arrow, 30 kDa band (glycosylated); black arrow, 25 kDa band (non-glycosylated). ( B ) Point mutants of human podoplanin were treated with NZ-1 and LpMab-3 (1 μg/mL) for 30 min at 4°C, followed by treatment with Alexa Fluor 488 conjugated anti-rat IgG and anti-mouse IgG, respectively. Fluorescence data were collected using a Cell Analyzer EC800. ( C ) TGIRIE sequence and α2–6 linked sialic acid are the critical epitope of LpMab-3.

    Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

    Article Title: Characterization of Monoclonal Antibody LpMab-3 Recognizing Sialylated Glycopeptide of Podoplanin

    doi: 10.1089/mab.2014.0087

    Figure Lengend Snippet: Epitope mapping of LpMab-7 by Western blot analysis and flow cytometry. ( A ) Western blotting by LpMab-3, LpMab-7, NZ-1, r2336, 1E6, RMab-3 (α-IDH1), and AC-15 (α-β-actin). Total cell lysate were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/mL of primary antibodies and then with peroxidase-conjugated secondary antibodies; the membrane was detected using a Sayaca-Imager. Blue arrow, 40 kDa band (glycosylated); red arrow, 30 kDa band (glycosylated); black arrow, 25 kDa band (non-glycosylated). ( B ) Point mutants of human podoplanin were treated with NZ-1 and LpMab-3 (1 μg/mL) for 30 min at 4°C, followed by treatment with Alexa Fluor 488 conjugated anti-rat IgG and anti-mouse IgG, respectively. Fluorescence data were collected using a Cell Analyzer EC800. ( C ) TGIRIE sequence and α2–6 linked sialic acid are the critical epitope of LpMab-3.

    Article Snippet: Flow cytometry Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Wako Pure Chemical Industries). After washing with phosphate-buffered saline (PBS), the cells were treated with primary antibodies (1 μg/mL) for 30 min at 4°C, followed by treatment with Oregon green-conjugated anti-mouse IgG (Life Technologies), Alexa Fluor 488 conjugated anti-mouse IgG (Cell Signaling Technology, Danvers, MA), or Alexa Fluor 488 conjugated anti-rat IgG (Cell Signaling Technology).

    Techniques: Western Blot, Flow Cytometry, Cytometry, Blocking Assay, Incubation, Fluorescence, Sequencing

    ( A ) Flow cytometric analysis by LpMab-3 against LN229/hPDPN and LN229. Cell lines were treated with LpMab-3 (1 μg/mL) for 30 min at 4°C, followed by treatment with Oregon green-conjugated anti-mouse IgG. Fluorescence data were collected using a FACS Calibur flow cytometer. ( B ) Western blot analysis by LpMab-3. Total cell lysate were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/mL of LpMab-3 and then with peroxidase-conjugated anti-mouse IgG; the membrane was detected using a Sayaca-Imager. ( C ) Flow cytometric analysis by LpMab-3 and LpMab-7 against glycan-deficient podoplanin-expressing CHO cell lines. Cell lines were treated with LpMab-3 and LpMab-7 (1 μg/mL) for 30 min at 4°C, followed by treatment with Alexa Fluor 488 conjugated anti-mouse IgG. Fluorescence data were collected using a Cell Analyzer EC800. ( D, E ) Immunohistochemical analysis against normal tissues using LpMab-3. Sections of normal lung ( D ) and normal colon ( E ) were incubated with 5 μg/mL of LpMab-3, followed by Envision+ kit. Color was developed using DAB and counterstained with hematoxylin.

    Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

    Article Title: Characterization of Monoclonal Antibody LpMab-3 Recognizing Sialylated Glycopeptide of Podoplanin

    doi: 10.1089/mab.2014.0087

    Figure Lengend Snippet: ( A ) Flow cytometric analysis by LpMab-3 against LN229/hPDPN and LN229. Cell lines were treated with LpMab-3 (1 μg/mL) for 30 min at 4°C, followed by treatment with Oregon green-conjugated anti-mouse IgG. Fluorescence data were collected using a FACS Calibur flow cytometer. ( B ) Western blot analysis by LpMab-3. Total cell lysate were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/mL of LpMab-3 and then with peroxidase-conjugated anti-mouse IgG; the membrane was detected using a Sayaca-Imager. ( C ) Flow cytometric analysis by LpMab-3 and LpMab-7 against glycan-deficient podoplanin-expressing CHO cell lines. Cell lines were treated with LpMab-3 and LpMab-7 (1 μg/mL) for 30 min at 4°C, followed by treatment with Alexa Fluor 488 conjugated anti-mouse IgG. Fluorescence data were collected using a Cell Analyzer EC800. ( D, E ) Immunohistochemical analysis against normal tissues using LpMab-3. Sections of normal lung ( D ) and normal colon ( E ) were incubated with 5 μg/mL of LpMab-3, followed by Envision+ kit. Color was developed using DAB and counterstained with hematoxylin.

    Article Snippet: Flow cytometry Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Wako Pure Chemical Industries). After washing with phosphate-buffered saline (PBS), the cells were treated with primary antibodies (1 μg/mL) for 30 min at 4°C, followed by treatment with Oregon green-conjugated anti-mouse IgG (Life Technologies), Alexa Fluor 488 conjugated anti-mouse IgG (Cell Signaling Technology, Danvers, MA), or Alexa Fluor 488 conjugated anti-rat IgG (Cell Signaling Technology).

    Techniques: Flow Cytometry, Fluorescence, FACS, Cytometry, Western Blot, Blocking Assay, Incubation, Expressing, Immunohistochemistry

    Autophagosomes originated from the ER but not mitochondria. a Marc-145 cells were transfected with Nsp3-mCherry and Nsp5-mCherry for 24 h; ATG5 is labeled in green, and nuclei were stained with DAPI (blue). Scale bars: 10 μm. Statistical analysis of the Pearson correlation coefficient for calnexin and ATG5. The Pearson colocalization coefficient is presented as the ratio of punctate signals of calnexin that were positive for ATG5. b Nsp3-mCherry and Nsp5-mCherry were expressed in Marc-145 cells. Twenty-four hours after transfection, cells were cultured with MitoTracker Green, a marker of mitochondria, and were then fixed and immunostained with an anti-ATG5 antibody (pink). Nuclei were stained with DAPI (blue). Scale bars: 10 μm. Statistical analysis of the Pearson correlation coefficient for MitoTracker Green and ATG5. The colocalization coefficient is presented as the ratio of fluorescent signals of MitoTracker Green that were negative for ATG5

    Journal: Virology Journal

    Article Title: Involvement of PRRSV NSP3 and NSP5 in the autophagy process

    doi: 10.1186/s12985-019-1116-x

    Figure Lengend Snippet: Autophagosomes originated from the ER but not mitochondria. a Marc-145 cells were transfected with Nsp3-mCherry and Nsp5-mCherry for 24 h; ATG5 is labeled in green, and nuclei were stained with DAPI (blue). Scale bars: 10 μm. Statistical analysis of the Pearson correlation coefficient for calnexin and ATG5. The Pearson colocalization coefficient is presented as the ratio of punctate signals of calnexin that were positive for ATG5. b Nsp3-mCherry and Nsp5-mCherry were expressed in Marc-145 cells. Twenty-four hours after transfection, cells were cultured with MitoTracker Green, a marker of mitochondria, and were then fixed and immunostained with an anti-ATG5 antibody (pink). Nuclei were stained with DAPI (blue). Scale bars: 10 μm. Statistical analysis of the Pearson correlation coefficient for MitoTracker Green and ATG5. The colocalization coefficient is presented as the ratio of fluorescent signals of MitoTracker Green that were negative for ATG5

    Article Snippet: Rabbit anti-LC3, anti-calnexin, and anti-GAPDH; mouse anti-ATG5; Alexa Fluor 488-conjugated anti-rabbit IgG; Alexa Fluor 488-conjugated anti-mouse IgG; and Alexa Fluor 647-conjugated anti-mouse IgG secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Transfection, Labeling, Staining, Cell Culture, Marker