rabbit anti ha tag mab  (Cell Signaling Technology Inc)

 
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    Name:
    HA Tag C29F4 Rabbit mAb Sepharose Bead Conjugate
    Description:
    This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N hydroxysuccinimide NHS activated Sepharose beads HA Tag C29F4 Rabbit mAb Sepharose Bead Conjugate is useful for the immunoprecipitation of HA tagged recombinant proteins
    Catalog Number:
    3956
    Price:
    None
    Category:
    WB IP Reagents
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide containing the influenza hemagglutinin epitope (YPYDVPDYA).
    Reactivity:
    All Species Expected
    Applications:
    Immunoprecipitation
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc rabbit anti ha tag mab
    Ccr1l1 encodes a transmembrane protein that traffics to the plasma membrane and contains an extracellular N terminus and an intracellular C terminus. A , schematic representation of the pNT1 and pNT2 constructs cloned into a pcDNA3.1 plasmid for the expression of Ccr1l1 with a C-terminal or N-terminal <t>HA</t> <t>tag,</t> respectively. Ccr1l1 ORF is indicated by a black box , whereas the HA tag (HA) and kozak sequence (K) are shown in white boxes . The position of the ATG initiation codon is indicated above each graph. The expression of these constructs was analyzed by fluorescence microscopy ( B ) and FACS ( C and D ) in HEK293-transfected cells. B , fluorescence microscopy images of the <t>anti-HA</t> ( green ) and DAPI ( blue ) staining of HEK293 cells transfected with the plasmids indicated on the left of each row with (+Triton) or without (–Triton) permeabilization, as indicated above each column (400× magnification, scale bar = 20 μm). C , FACS histograms of the anti-HA staining of permeabilized or nonpermeabilized (as indicated above each graph) HEK293 cells transfected with the plasmids indicated in the insets of the right graph. These results are quantified in panel D . Bars represent the mean ± SD median fluorescence intensity (MFI) of three independent transfections from one experiment representative of three independent experiments. p values from a one-way ANOVA with Tukey correction for multiple comparisons are indicated.
    This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N hydroxysuccinimide NHS activated Sepharose beads HA Tag C29F4 Rabbit mAb Sepharose Bead Conjugate is useful for the immunoprecipitation of HA tagged recombinant proteins
    https://www.bioz.com/result/rabbit anti ha tag mab/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti ha tag mab - by Bioz Stars, 2021-09
    95/100 stars

    Images

    1) Product Images from "Structural and functional analysis of Ccr1l1, a Rodentia-restricted eosinophil-selective chemokine receptor homologue"

    Article Title: Structural and functional analysis of Ccr1l1, a Rodentia-restricted eosinophil-selective chemokine receptor homologue

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.100373

    Ccr1l1 encodes a transmembrane protein that traffics to the plasma membrane and contains an extracellular N terminus and an intracellular C terminus. A , schematic representation of the pNT1 and pNT2 constructs cloned into a pcDNA3.1 plasmid for the expression of Ccr1l1 with a C-terminal or N-terminal HA tag, respectively. Ccr1l1 ORF is indicated by a black box , whereas the HA tag (HA) and kozak sequence (K) are shown in white boxes . The position of the ATG initiation codon is indicated above each graph. The expression of these constructs was analyzed by fluorescence microscopy ( B ) and FACS ( C and D ) in HEK293-transfected cells. B , fluorescence microscopy images of the anti-HA ( green ) and DAPI ( blue ) staining of HEK293 cells transfected with the plasmids indicated on the left of each row with (+Triton) or without (–Triton) permeabilization, as indicated above each column (400× magnification, scale bar = 20 μm). C , FACS histograms of the anti-HA staining of permeabilized or nonpermeabilized (as indicated above each graph) HEK293 cells transfected with the plasmids indicated in the insets of the right graph. These results are quantified in panel D . Bars represent the mean ± SD median fluorescence intensity (MFI) of three independent transfections from one experiment representative of three independent experiments. p values from a one-way ANOVA with Tukey correction for multiple comparisons are indicated.
    Figure Legend Snippet: Ccr1l1 encodes a transmembrane protein that traffics to the plasma membrane and contains an extracellular N terminus and an intracellular C terminus. A , schematic representation of the pNT1 and pNT2 constructs cloned into a pcDNA3.1 plasmid for the expression of Ccr1l1 with a C-terminal or N-terminal HA tag, respectively. Ccr1l1 ORF is indicated by a black box , whereas the HA tag (HA) and kozak sequence (K) are shown in white boxes . The position of the ATG initiation codon is indicated above each graph. The expression of these constructs was analyzed by fluorescence microscopy ( B ) and FACS ( C and D ) in HEK293-transfected cells. B , fluorescence microscopy images of the anti-HA ( green ) and DAPI ( blue ) staining of HEK293 cells transfected with the plasmids indicated on the left of each row with (+Triton) or without (–Triton) permeabilization, as indicated above each column (400× magnification, scale bar = 20 μm). C , FACS histograms of the anti-HA staining of permeabilized or nonpermeabilized (as indicated above each graph) HEK293 cells transfected with the plasmids indicated in the insets of the right graph. These results are quantified in panel D . Bars represent the mean ± SD median fluorescence intensity (MFI) of three independent transfections from one experiment representative of three independent experiments. p values from a one-way ANOVA with Tukey correction for multiple comparisons are indicated.

    Techniques Used: Construct, Clone Assay, Plasmid Preparation, Expressing, Sequencing, Fluorescence, Microscopy, FACS, Transfection, Staining

    Related Articles

    Transfection:

    Article Title: Interaction of dengue NS3 with human RNA silencing machinery through HSPA1A
    Article Snippet: .. Forty eight hours post transfection, the cells were lysed in non-denaturing conditions and co-immunoprecipitation was performed using HA-Tag Rabbit mAb Sepharose Bead Conjugate (Cell Signalling Technology Inc., USA). ..

    Article Title: Bcl-2-associated athanogene 5 (BAG5) regulates Parkin-dependent mitophagy and cell death
    Article Snippet: .. HA-tag immunoprecipitation SH-SY5Y cells stably overexpressing GFP or GFP-BAG5 were transfected with either siNTC or siParkin plus HA-Ubiquitin and then treated with 50 μm CCCP for 18 h. Lysates from these cells were incubated overnight at 4 °C with anti-HA-tag antibody conjugated sepharose beads (Cell Signaling Technology 3956). ..

    Incubation:

    Article Title: Thalamo-hippocampal pathway regulates incidental memory load: insights from sex differences
    Article Snippet: .. For DREADDs experiments, after rinsing in 1× PBS, brain sections were incubated in blocking solution made of NGS 5% and 0.3% Triton X-100 in PBS for 1 h. Blocking solution was then removed and replaced with primary antibody solution made of NGS 1%, Triton X-100 0.3% and rabbit anti-HA tag (#3724; Cell Signaling) diluted 1:500 overnight at 4°C. ..

    Article Title: Bcl-2-associated athanogene 5 (BAG5) regulates Parkin-dependent mitophagy and cell death
    Article Snippet: .. HA-tag immunoprecipitation SH-SY5Y cells stably overexpressing GFP or GFP-BAG5 were transfected with either siNTC or siParkin plus HA-Ubiquitin and then treated with 50 μm CCCP for 18 h. Lysates from these cells were incubated overnight at 4 °C with anti-HA-tag antibody conjugated sepharose beads (Cell Signaling Technology 3956). ..

    Blocking Assay:

    Article Title: Thalamo-hippocampal pathway regulates incidental memory load: insights from sex differences
    Article Snippet: .. For DREADDs experiments, after rinsing in 1× PBS, brain sections were incubated in blocking solution made of NGS 5% and 0.3% Triton X-100 in PBS for 1 h. Blocking solution was then removed and replaced with primary antibody solution made of NGS 1%, Triton X-100 0.3% and rabbit anti-HA tag (#3724; Cell Signaling) diluted 1:500 overnight at 4°C. ..

    Next-Generation Sequencing:

    Article Title: Thalamo-hippocampal pathway regulates incidental memory load: insights from sex differences
    Article Snippet: .. For DREADDs experiments, after rinsing in 1× PBS, brain sections were incubated in blocking solution made of NGS 5% and 0.3% Triton X-100 in PBS for 1 h. Blocking solution was then removed and replaced with primary antibody solution made of NGS 1%, Triton X-100 0.3% and rabbit anti-HA tag (#3724; Cell Signaling) diluted 1:500 overnight at 4°C. ..

    Staining:

    Article Title: Structural and functional analysis of Ccr1l1, a Rodentia-restricted eosinophil-selective chemokine receptor homologue
    Article Snippet: .. Then, 400,000 cells were stained with Zombie Violet viability dye (Biolegend) for 10 min at room temperature and a rabbit anti-HA tag mAb (Cell Signaling Technology, Danvers, MA) on ice for 20 min followed by an anti-rabbit F(ab′)2 fragment conjugated with AlexaFluor 488 (Cell Signaling Technology). ..

    Article Title: ZHX2 Promotes HIF1α Oncogenic Signaling in Triple-Negative Breast Cancer
    Article Snippet: .. AntibodiesAntibodies used for immunoblotting, immunoprecipitation and IHC staining were as follows: Rabbit anti ZHX2 antibody (Genetex, 112232), Rabbit anti HIF1α (Cell Signaling, 36169), Rabbit anti HIF1β (Cell Signaling, 5537), Rabbit anti VHL (Cell Signaling, 68547), rabbit anti HA tag (Cell Signaling, 3724), mouse anti α-Tubulin (Cell Signaling, 3873). ..

    Co-Immunoprecipitation Assay:

    Article Title: TgROP18 targets IL20RB for host-defense-related-STAT3 activation during Toxoplasma gondii infection
    Article Snippet: .. The antibody used for IP or Co-IP: rabbit anti-HA tag (4 μg) was purchased from Cell Signaling Technology. ..

    Concentration Assay:

    Article Title: Distributed control of motor circuits for backward walking in Drosophila
    Article Snippet: .. The antibodies and dyes used were Cy2 SNAP-tag ligand (Luke Lavis, JRC, 10 μL/mL for final concentration of 2 μM), primary rat anti-FLAG Tag (Novus Biologicals NBP1-06712, 1:200), primary rabbit anti-HA Tag (Cell Signal Technologies 3724 S, 1:300), secondary ATTO647N goat anti-rat (Rockland 612-156-120, 1:300), secondary AF594 donkey anti-rabbit (Jackson ImmunoResearch 711-585-152, 1:500), and DL550 mouse anti-V5 (AbD Serotec MCA1360D550GA, 1:500). ..

    Immunoprecipitation:

    Article Title: ZHX2 Promotes HIF1α Oncogenic Signaling in Triple-Negative Breast Cancer
    Article Snippet: .. AntibodiesAntibodies used for immunoblotting, immunoprecipitation and IHC staining were as follows: Rabbit anti ZHX2 antibody (Genetex, 112232), Rabbit anti HIF1α (Cell Signaling, 36169), Rabbit anti HIF1β (Cell Signaling, 5537), Rabbit anti VHL (Cell Signaling, 68547), rabbit anti HA tag (Cell Signaling, 3724), mouse anti α-Tubulin (Cell Signaling, 3873). ..

    Article Title: Bcl-2-associated athanogene 5 (BAG5) regulates Parkin-dependent mitophagy and cell death
    Article Snippet: .. HA-tag immunoprecipitation SH-SY5Y cells stably overexpressing GFP or GFP-BAG5 were transfected with either siNTC or siParkin plus HA-Ubiquitin and then treated with 50 μm CCCP for 18 h. Lysates from these cells were incubated overnight at 4 °C with anti-HA-tag antibody conjugated sepharose beads (Cell Signaling Technology 3956). ..

    Immunohistochemistry:

    Article Title: ZHX2 Promotes HIF1α Oncogenic Signaling in Triple-Negative Breast Cancer
    Article Snippet: .. AntibodiesAntibodies used for immunoblotting, immunoprecipitation and IHC staining were as follows: Rabbit anti ZHX2 antibody (Genetex, 112232), Rabbit anti HIF1α (Cell Signaling, 36169), Rabbit anti HIF1β (Cell Signaling, 5537), Rabbit anti VHL (Cell Signaling, 68547), rabbit anti HA tag (Cell Signaling, 3724), mouse anti α-Tubulin (Cell Signaling, 3873). ..

    Stable Transfection:

    Article Title: Bcl-2-associated athanogene 5 (BAG5) regulates Parkin-dependent mitophagy and cell death
    Article Snippet: .. HA-tag immunoprecipitation SH-SY5Y cells stably overexpressing GFP or GFP-BAG5 were transfected with either siNTC or siParkin plus HA-Ubiquitin and then treated with 50 μm CCCP for 18 h. Lysates from these cells were incubated overnight at 4 °C with anti-HA-tag antibody conjugated sepharose beads (Cell Signaling Technology 3956). ..

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    Cell Signaling Technology Inc rabbit anti ha monoclonal antibody
    LET-413 preferentially associated with the active RAB-5 and the inactive RAB-10. (A) let-413 open reading frames encoding let-413a , let-413b , and let-413c isoforms. Red arrowheads above let-413c indicate three single guide RNAs target locations in let-413(ycx23) CRISPR/Cas9 intestine somatic mutants. (B) LET-413c contains N-terminal LRRs and a C-terminal PDZ domain; amino acid numbers are indicated. (C) Glutathione beads loaded with GST and GST-LET-413 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-10(Q68L), RAB-8(Q67L), RAB-11(Q70L), RAB-35(Q69L), and ARF-6(Q67L) and inactive RAB-5(S33N), RAB-10(T23N), RAB-8(T22N), RAB-11(S25N), RAB-35(S24N), and ARF-6(T27N). Eluted proteins were separated on SDS-PAGE gel and analyzed by Western blotting using <t>anti–HA</t> antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (10%). (D) The binding affinity of GST-LET-413 was fourfold higher for RAB-5(Q78L) than for RAB-5(S33N). The SEMs from three independent experiments are shown. Asterisks indicate significant differences (**, P
    Rabbit Anti Ha Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti ha monoclonal antibody/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti ha monoclonal antibody - by Bioz Stars, 2021-09
    99/100 stars
      Buy from Supplier

    95
    Cell Signaling Technology Inc rabbit anti ha tag mab
    Ccr1l1 encodes a transmembrane protein that traffics to the plasma membrane and contains an extracellular N terminus and an intracellular C terminus. A , schematic representation of the pNT1 and pNT2 constructs cloned into a pcDNA3.1 plasmid for the expression of Ccr1l1 with a C-terminal or N-terminal <t>HA</t> <t>tag,</t> respectively. Ccr1l1 ORF is indicated by a black box , whereas the HA tag (HA) and kozak sequence (K) are shown in white boxes . The position of the ATG initiation codon is indicated above each graph. The expression of these constructs was analyzed by fluorescence microscopy ( B ) and FACS ( C and D ) in HEK293-transfected cells. B , fluorescence microscopy images of the <t>anti-HA</t> ( green ) and DAPI ( blue ) staining of HEK293 cells transfected with the plasmids indicated on the left of each row with (+Triton) or without (–Triton) permeabilization, as indicated above each column (400× magnification, scale bar = 20 μm). C , FACS histograms of the anti-HA staining of permeabilized or nonpermeabilized (as indicated above each graph) HEK293 cells transfected with the plasmids indicated in the insets of the right graph. These results are quantified in panel D . Bars represent the mean ± SD median fluorescence intensity (MFI) of three independent transfections from one experiment representative of three independent experiments. p values from a one-way ANOVA with Tukey correction for multiple comparisons are indicated.
    Rabbit Anti Ha Tag Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti ha tag mab/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti ha tag mab - by Bioz Stars, 2021-09
    95/100 stars
      Buy from Supplier

    Image Search Results


    LET-413 preferentially associated with the active RAB-5 and the inactive RAB-10. (A) let-413 open reading frames encoding let-413a , let-413b , and let-413c isoforms. Red arrowheads above let-413c indicate three single guide RNAs target locations in let-413(ycx23) CRISPR/Cas9 intestine somatic mutants. (B) LET-413c contains N-terminal LRRs and a C-terminal PDZ domain; amino acid numbers are indicated. (C) Glutathione beads loaded with GST and GST-LET-413 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-10(Q68L), RAB-8(Q67L), RAB-11(Q70L), RAB-35(Q69L), and ARF-6(Q67L) and inactive RAB-5(S33N), RAB-10(T23N), RAB-8(T22N), RAB-11(S25N), RAB-35(S24N), and ARF-6(T27N). Eluted proteins were separated on SDS-PAGE gel and analyzed by Western blotting using anti–HA antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (10%). (D) The binding affinity of GST-LET-413 was fourfold higher for RAB-5(Q78L) than for RAB-5(S33N). The SEMs from three independent experiments are shown. Asterisks indicate significant differences (**, P

    Journal: The Journal of Cell Biology

    Article Title: LET-413/Erbin acts as a RAB-5 effector to promote RAB-10 activation during endocytic recycling

    doi: 10.1083/jcb.201705136

    Figure Lengend Snippet: LET-413 preferentially associated with the active RAB-5 and the inactive RAB-10. (A) let-413 open reading frames encoding let-413a , let-413b , and let-413c isoforms. Red arrowheads above let-413c indicate three single guide RNAs target locations in let-413(ycx23) CRISPR/Cas9 intestine somatic mutants. (B) LET-413c contains N-terminal LRRs and a C-terminal PDZ domain; amino acid numbers are indicated. (C) Glutathione beads loaded with GST and GST-LET-413 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-10(Q68L), RAB-8(Q67L), RAB-11(Q70L), RAB-35(Q69L), and ARF-6(Q67L) and inactive RAB-5(S33N), RAB-10(T23N), RAB-8(T22N), RAB-11(S25N), RAB-35(S24N), and ARF-6(T27N). Eluted proteins were separated on SDS-PAGE gel and analyzed by Western blotting using anti–HA antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (10%). (D) The binding affinity of GST-LET-413 was fourfold higher for RAB-5(Q78L) than for RAB-5(S33N). The SEMs from three independent experiments are shown. Asterisks indicate significant differences (**, P

    Article Snippet: Antibodies The antibodies used in this study were rabbit anti–actin polyclonal antibody (sc-1616-R; Santa Cruz Biotechnology), rabbit anti–HA monoclonal antibody (C29F4; Cell Signaling Technology), rabbit anti–GST monoclonal antibody (91G1; Cell Signaling Technology), rabbit anti–GFP polyclonal antibody ChIP grade (ab290; Abcam), rabbit anti–Erbin polyclonal antibody (ab55930; Abcam), rabbit anti–Rab5 monoclonal antibody (3547; Cell Signaling Technology), and anti–rabbit IgG-Alexa Fluor 594 conjugate (8889; Cell Signaling Technology).

    Techniques: CRISPR, Incubation, In Vitro, SDS Page, Western Blot, Binding Assay

    LET-413 preferentially associated with the active RAB-5 and the inactive RAB-10. (A) let-413 open reading frames encoding let-413a , let-413b , and let-413c isoforms. Red arrowheads above let-413c indicate three single guide RNAs target locations in let-413(ycx23) CRISPR/Cas9 intestine somatic mutants. (B) LET-413c contains N-terminal LRRs and a C-terminal PDZ domain; amino acid numbers are indicated. (C) Glutathione beads loaded with GST and GST-LET-413 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-10(Q68L), RAB-8(Q67L), RAB-11(Q70L), RAB-35(Q69L), and ARF-6(Q67L) and inactive RAB-5(S33N), RAB-10(T23N), RAB-8(T22N), RAB-11(S25N), RAB-35(S24N), and ARF-6(T27N). Eluted proteins were separated on SDS-PAGE gel and analyzed by Western blotting using anti–HA antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (10%). (D) The binding affinity of GST-LET-413 was fourfold higher for RAB-5(Q78L) than for RAB-5(S33N). The SEMs from three independent experiments are shown. Asterisks indicate significant differences (**, P

    Journal: The Journal of Cell Biology

    Article Title: LET-413/Erbin acts as a RAB-5 effector to promote RAB-10 activation during endocytic recycling

    doi: 10.1083/jcb.201705136

    Figure Lengend Snippet: LET-413 preferentially associated with the active RAB-5 and the inactive RAB-10. (A) let-413 open reading frames encoding let-413a , let-413b , and let-413c isoforms. Red arrowheads above let-413c indicate three single guide RNAs target locations in let-413(ycx23) CRISPR/Cas9 intestine somatic mutants. (B) LET-413c contains N-terminal LRRs and a C-terminal PDZ domain; amino acid numbers are indicated. (C) Glutathione beads loaded with GST and GST-LET-413 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-10(Q68L), RAB-8(Q67L), RAB-11(Q70L), RAB-35(Q69L), and ARF-6(Q67L) and inactive RAB-5(S33N), RAB-10(T23N), RAB-8(T22N), RAB-11(S25N), RAB-35(S24N), and ARF-6(T27N). Eluted proteins were separated on SDS-PAGE gel and analyzed by Western blotting using anti–HA antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (10%). (D) The binding affinity of GST-LET-413 was fourfold higher for RAB-5(Q78L) than for RAB-5(S33N). The SEMs from three independent experiments are shown. Asterisks indicate significant differences (**, P

    Article Snippet: The antibodies used in this study were rabbit anti–actin polyclonal antibody (sc-1616-R; Santa Cruz Biotechnology), rabbit anti–HA monoclonal antibody (C29F4; Cell Signaling Technology), rabbit anti–GST monoclonal antibody (91G1; Cell Signaling Technology), rabbit anti–GFP polyclonal antibody ChIP grade (ab290; Abcam), rabbit anti–Erbin polyclonal antibody (ab55930; Abcam), rabbit anti–Rab5 monoclonal antibody (3547; Cell Signaling Technology), and anti–rabbit IgG-Alexa Fluor 594 conjugate (8889; Cell Signaling Technology).

    Techniques: CRISPR, Incubation, In Vitro, SDS Page, Western Blot, Binding Assay

    SAC-1 interacts with the active and inactive mutant forms of ARF-6, and the residence of SAC-1 in basolateral puncta requires ARF-6. (A) Western blot showing GST pull-down with in vitro–translated HA-tagged proteins. Glutathione beads loaded with GST and GST-SAC-1 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-7(Q68L), RAB-8(Q67L), RAB-10(Q68L), RAB-11(Q70L), ARF-6(Q67L), and ARF-6(T27N). Eluted proteins were separated on the SDS-PAGE gel and analyzed by Western blotting using anti–HA antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (5%). (B and B′) SAC-1 colocalized well with ARF-6–, RAB-5–, or RAB-10–labeled endosomes. In addition, SAC-1 overlaps significantly with the TGN marker AMAN-2 and the ER marker SP12. Nevertheless, SAC-1 displayed little colocalization with the late endosome marker RAB-7. Arrowheads indicate positive overlap. Pearson’s correlation coefficients for GFP and mCherry signals were calculated ( n = 6 animals). (C and C′) Compared with wild-type animals, the labeling of GFP-SAC-1 in basolateral puncta was significantly reduced in the absence of ARF-6 (top focal plane). Transgenic expression of ARF-6(Q67L)-mCherry rescued the puncta labeling GFP-SAC-1, and the transgenic expression of ARF-6(T27N)-mCherry failed to restore the endosomal localization of GFP-SAC-1. Asterisks in the panels indicate intestinal lumen. Error bars represent SEM ( n = 18 each), and asterisks indicate the significant differences in the one-tailed Student’s t test (ns, no significance; **, P

    Journal: The Journal of Cell Biology

    Article Title: SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity

    doi: 10.1083/jcb.201711065

    Figure Lengend Snippet: SAC-1 interacts with the active and inactive mutant forms of ARF-6, and the residence of SAC-1 in basolateral puncta requires ARF-6. (A) Western blot showing GST pull-down with in vitro–translated HA-tagged proteins. Glutathione beads loaded with GST and GST-SAC-1 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-7(Q68L), RAB-8(Q67L), RAB-10(Q68L), RAB-11(Q70L), ARF-6(Q67L), and ARF-6(T27N). Eluted proteins were separated on the SDS-PAGE gel and analyzed by Western blotting using anti–HA antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (5%). (B and B′) SAC-1 colocalized well with ARF-6–, RAB-5–, or RAB-10–labeled endosomes. In addition, SAC-1 overlaps significantly with the TGN marker AMAN-2 and the ER marker SP12. Nevertheless, SAC-1 displayed little colocalization with the late endosome marker RAB-7. Arrowheads indicate positive overlap. Pearson’s correlation coefficients for GFP and mCherry signals were calculated ( n = 6 animals). (C and C′) Compared with wild-type animals, the labeling of GFP-SAC-1 in basolateral puncta was significantly reduced in the absence of ARF-6 (top focal plane). Transgenic expression of ARF-6(Q67L)-mCherry rescued the puncta labeling GFP-SAC-1, and the transgenic expression of ARF-6(T27N)-mCherry failed to restore the endosomal localization of GFP-SAC-1. Asterisks in the panels indicate intestinal lumen. Error bars represent SEM ( n = 18 each), and asterisks indicate the significant differences in the one-tailed Student’s t test (ns, no significance; **, P

    Article Snippet: Antibodies The antibodies used in the current study are listed below: rabbit anti–actin polyclonal antibody (sc-1616-R; Santa Cruz Biotechnologies), rabbit anti–HA monoclonal antibody (C29F4; Cell Signaling Technology), rabbit anti–GST monoclonal antibody (91G1; Cell Signaling Technology), rabbit anti–mCherry polyclonal antibody (ab167453; Abcam), rabbit anti–GFP polyclonal antibody–chromatin immunoprecipitation grade (ab290; Abcam), mouse anti–P(4,5)P2 monoclonal antibody (Z-P045; Echelon Biosciences), and mouse anti–PIPn monoclonal antibody (Z-P999; Echelon Biosciences).

    Techniques: Mutagenesis, Western Blot, In Vitro, Incubation, SDS Page, Binding Assay, Labeling, Marker, Transgenic Assay, Expressing, One-tailed Test

    Ccr1l1 encodes a transmembrane protein that traffics to the plasma membrane and contains an extracellular N terminus and an intracellular C terminus. A , schematic representation of the pNT1 and pNT2 constructs cloned into a pcDNA3.1 plasmid for the expression of Ccr1l1 with a C-terminal or N-terminal HA tag, respectively. Ccr1l1 ORF is indicated by a black box , whereas the HA tag (HA) and kozak sequence (K) are shown in white boxes . The position of the ATG initiation codon is indicated above each graph. The expression of these constructs was analyzed by fluorescence microscopy ( B ) and FACS ( C and D ) in HEK293-transfected cells. B , fluorescence microscopy images of the anti-HA ( green ) and DAPI ( blue ) staining of HEK293 cells transfected with the plasmids indicated on the left of each row with (+Triton) or without (–Triton) permeabilization, as indicated above each column (400× magnification, scale bar = 20 μm). C , FACS histograms of the anti-HA staining of permeabilized or nonpermeabilized (as indicated above each graph) HEK293 cells transfected with the plasmids indicated in the insets of the right graph. These results are quantified in panel D . Bars represent the mean ± SD median fluorescence intensity (MFI) of three independent transfections from one experiment representative of three independent experiments. p values from a one-way ANOVA with Tukey correction for multiple comparisons are indicated.

    Journal: The Journal of Biological Chemistry

    Article Title: Structural and functional analysis of Ccr1l1, a Rodentia-restricted eosinophil-selective chemokine receptor homologue

    doi: 10.1016/j.jbc.2021.100373

    Figure Lengend Snippet: Ccr1l1 encodes a transmembrane protein that traffics to the plasma membrane and contains an extracellular N terminus and an intracellular C terminus. A , schematic representation of the pNT1 and pNT2 constructs cloned into a pcDNA3.1 plasmid for the expression of Ccr1l1 with a C-terminal or N-terminal HA tag, respectively. Ccr1l1 ORF is indicated by a black box , whereas the HA tag (HA) and kozak sequence (K) are shown in white boxes . The position of the ATG initiation codon is indicated above each graph. The expression of these constructs was analyzed by fluorescence microscopy ( B ) and FACS ( C and D ) in HEK293-transfected cells. B , fluorescence microscopy images of the anti-HA ( green ) and DAPI ( blue ) staining of HEK293 cells transfected with the plasmids indicated on the left of each row with (+Triton) or without (–Triton) permeabilization, as indicated above each column (400× magnification, scale bar = 20 μm). C , FACS histograms of the anti-HA staining of permeabilized or nonpermeabilized (as indicated above each graph) HEK293 cells transfected with the plasmids indicated in the insets of the right graph. These results are quantified in panel D . Bars represent the mean ± SD median fluorescence intensity (MFI) of three independent transfections from one experiment representative of three independent experiments. p values from a one-way ANOVA with Tukey correction for multiple comparisons are indicated.

    Article Snippet: Then, 400,000 cells were stained with Zombie Violet viability dye (Biolegend) for 10 min at room temperature and a rabbit anti-HA tag mAb (Cell Signaling Technology, Danvers, MA) on ice for 20 min followed by an anti-rabbit F(ab′)2 fragment conjugated with AlexaFluor 488 (Cell Signaling Technology).

    Techniques: Construct, Clone Assay, Plasmid Preparation, Expressing, Sequencing, Fluorescence, Microscopy, FACS, Transfection, Staining