rabbit anti ha monoclonal antibody  (Cell Signaling Technology Inc)

 
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    Name:
    HA Tag C29F4 Rabbit mAb
    Description:
    Epitope tags are useful for the labeling and detection of proteins using immunoblotting immunoprecipitation and immunostaining techniques Because of their small size they are unlikely to affect the tagged protein s biochemical properties The HA tag is derived from an epitope of the influenza hemagglutinin protein which has been used extensively as a general epitope tag in expression vectors 1
    Catalog Number:
    3724
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide containing the influenza hemagglutinin epitope (YPYDVPDYA).
    Reactivity:
    All Species Expected
    Applications:
    Western Blot, Immunoprecipitation, Immunohistochemistry, Immunofluorescence, Flow Cytometry, Chromatin Immunoprecipitation
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    Structured Review

    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
    Epitope tags are useful for the labeling and detection of proteins using immunoblotting immunoprecipitation and immunostaining techniques Because of their small size they are unlikely to affect the tagged protein s biochemical properties The HA tag is derived from an epitope of the influenza hemagglutinin protein which has been used extensively as a general epitope tag in expression vectors 1
    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

    Images

    1) Product Images from "LET-413/Erbin acts as a RAB-5 effector to promote RAB-10 activation during endocytic recycling"

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

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201705136

    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
    Figure Legend 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

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

    2) Product Images from "LET-413/Erbin acts as a RAB-5 effector to promote RAB-10 activation during endocytic recycling"

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

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201705136

    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
    Figure Legend 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

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

    3) Product Images from "SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity"

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

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201711065

    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
    Figure Legend 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

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

    4) Product Images from "SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity"

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

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201711065

    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
    Figure Legend 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

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

    5) Product Images from "Establishment of a tagged variant of Lgr4 receptor suitable for functional and expression studies in the mouse"

    Article Title: Establishment of a tagged variant of Lgr4 receptor suitable for functional and expression studies in the mouse

    Journal: Transgenic Research

    doi: 10.1007/s11248-017-0027-0

    Generation of mice harboring a modified Lgr4 allele producing the 3HA–Lgr4 fusion protein. a The top diagram shows TALEN-based genome editing of the Lgr4 locus; exons are depicted by black boxes . A pair of TALENs (scissors pictogram) recognizes and cleaves a specific sequence in the first exon of the gene. The affected locus is repaired by homologous recombination using an exogenous template carrying a portion of the Lgr4 gene including the 3HA tag sequence ( red box ). Correct targeting (at both ends of the template) is verified by sequencing of PCR products amplified from genomic DNA using two primer pairs: P1 and P2, P3 and P4; primer positions are depicted by black arrows . Notice that one primer from each set, i.e., P1 and P4, primes in a sequence that is not present in the targeting construct. For regular genotyping, i.e., upon establishment of Lgr4 +/3HA and Lgr4 3HA/3HA strains, PCR with P5 and P6 primers ( red arrows ) generating shorter DNA fragments were utilized. b Top, TALEN recognition sites (sequence in red ) in the first Lgr4 exon; translational start is boxed in red . The numbers above the sequence indicate nucleotide positions in the mouse genome assembly GRCm38:CM000995.2 (nucleotide numbering in other schemes was also taken from the same genome assembly). Middle the nucleotide sequence of the corresponding part of the Lgr4 locus upon insertion of the 3HA tag encoding sequence. Bottom the amino acid sequence of Lgr4 N-terminus fused to 3HA tag. c Agarose gel electrophoresis discriminating Lgr4 +/+ (WT/WT), Lgr4 +/3HA (WT/3HA), and Lgr4 3HA/3HA (3HA/3HA) animals using P1 and P2 ( left gel), and P5 and P6 ( right gel) primers. The gel containing DNA fragments obtained by PCR with primers P3 and P4 that are hardly distinguishable by standard electrophoresis (fragment sizes 1462 bp for wt and 1555 bp for the targeted allele) is not shown. d Cell-based functionality test of the tagged LGR4 receptor. Immunofluorescent microscopy images of HeLa cells transfected with the construct expressing human LGR4 protein tagged with the 3HA and FLAG tag at its N- and C-terminus, respectively. A rabbit monoclonal antibody recognizing the 3HA tag or mouse anti-FLAG antibodies were incubated either with living ( left image) or fixed (and permeabilized) cells ( right image) grown on coverslips. The retained immunoglobulins were visualized by fluorescently labeled goat anti-rabbit ( red fluorescence) or mouse anti-FLAG tag ( green fluorescence) secondary antibodies. Notice that whereas both antibodies stained fixed cells, living cells were recognized only with the 3HA-specific antibody reacting with the extracellular portion of 3HA–LGR4–FLAG fusion protein (see also the schemes on the left sides of the images). (Color figure online)
    Figure Legend Snippet: Generation of mice harboring a modified Lgr4 allele producing the 3HA–Lgr4 fusion protein. a The top diagram shows TALEN-based genome editing of the Lgr4 locus; exons are depicted by black boxes . A pair of TALENs (scissors pictogram) recognizes and cleaves a specific sequence in the first exon of the gene. The affected locus is repaired by homologous recombination using an exogenous template carrying a portion of the Lgr4 gene including the 3HA tag sequence ( red box ). Correct targeting (at both ends of the template) is verified by sequencing of PCR products amplified from genomic DNA using two primer pairs: P1 and P2, P3 and P4; primer positions are depicted by black arrows . Notice that one primer from each set, i.e., P1 and P4, primes in a sequence that is not present in the targeting construct. For regular genotyping, i.e., upon establishment of Lgr4 +/3HA and Lgr4 3HA/3HA strains, PCR with P5 and P6 primers ( red arrows ) generating shorter DNA fragments were utilized. b Top, TALEN recognition sites (sequence in red ) in the first Lgr4 exon; translational start is boxed in red . The numbers above the sequence indicate nucleotide positions in the mouse genome assembly GRCm38:CM000995.2 (nucleotide numbering in other schemes was also taken from the same genome assembly). Middle the nucleotide sequence of the corresponding part of the Lgr4 locus upon insertion of the 3HA tag encoding sequence. Bottom the amino acid sequence of Lgr4 N-terminus fused to 3HA tag. c Agarose gel electrophoresis discriminating Lgr4 +/+ (WT/WT), Lgr4 +/3HA (WT/3HA), and Lgr4 3HA/3HA (3HA/3HA) animals using P1 and P2 ( left gel), and P5 and P6 ( right gel) primers. The gel containing DNA fragments obtained by PCR with primers P3 and P4 that are hardly distinguishable by standard electrophoresis (fragment sizes 1462 bp for wt and 1555 bp for the targeted allele) is not shown. d Cell-based functionality test of the tagged LGR4 receptor. Immunofluorescent microscopy images of HeLa cells transfected with the construct expressing human LGR4 protein tagged with the 3HA and FLAG tag at its N- and C-terminus, respectively. A rabbit monoclonal antibody recognizing the 3HA tag or mouse anti-FLAG antibodies were incubated either with living ( left image) or fixed (and permeabilized) cells ( right image) grown on coverslips. The retained immunoglobulins were visualized by fluorescently labeled goat anti-rabbit ( red fluorescence) or mouse anti-FLAG tag ( green fluorescence) secondary antibodies. Notice that whereas both antibodies stained fixed cells, living cells were recognized only with the 3HA-specific antibody reacting with the extracellular portion of 3HA–LGR4–FLAG fusion protein (see also the schemes on the left sides of the images). (Color figure online)

    Techniques Used: Mouse Assay, Modification, TALENs, Sequencing, Homologous Recombination, Polymerase Chain Reaction, Amplification, Construct, Agarose Gel Electrophoresis, Electrophoresis, Microscopy, Transfection, Expressing, FLAG-tag, Incubation, Labeling, Fluorescence, Staining

    Related Articles

    Purification:

    Article Title: Long non-coding RNA KIKAT/LINC01061 as a novel epigenetic regulator that relocates KDM4A on chromatin and modulates viral reactivation
    Article Snippet: .. Primary antibodies against KDM4A (Polyclonal antibody purified from rabbit) [ ], K-bZIP (1:1000; Santa Cruz Biotechnology, sc-69797), Orf45 (1:1000; Santa Cruz Biotechnology, sc-53883), HA tag (1:4000; Cell Signaling Technology, #3724) and α-Tubulin (1:4000; Sigma, T6074-200UL) were used in this study. ..

    Article Title: The Transcription Factor PfAP2-O Influences Virulence Gene Transcription and Sexual Development in Plasmodium falciparum
    Article Snippet: .. Pull-down of proteins associated with AP2-O_GFP_HA_DD24 A total protein extract from the NF54::AP2-O_GFP_HA_DD24 clone was prepared and the protein complex containing the HA tag was purified using the HA-Tag (C29F4) Rabbit mAb (Sepharose Beads conjugate, Cell Signaling Technology) kit. ..

    Immunofluorescence:

    Article Title: Mapping the sequence specificity of heterotypic amyloid interactions enables the identification of aggregation modifiers
    Article Snippet: .. For immunofluorescence staining, primary antibody staining at 1:1000 dilution was performed in 1% BSA with an HA-tag (C29F4) Rabbit mAb (Cell Signalling, #3724), followed by secondary staining with an Alexa Fluor 647 goat anti-rabbit antibody (ThermoFisher, A-21245) at 1:1000 dilution in 1% BSA for 1h. ..

    Staining:

    Article Title: Mapping the sequence specificity of heterotypic amyloid interactions enables the identification of aggregation modifiers
    Article Snippet: .. For immunofluorescence staining, primary antibody staining at 1:1000 dilution was performed in 1% BSA with an HA-tag (C29F4) Rabbit mAb (Cell Signalling, #3724), followed by secondary staining with an Alexa Fluor 647 goat anti-rabbit antibody (ThermoFisher, A-21245) at 1:1000 dilution in 1% BSA for 1h. ..

    Western Blot:

    Article Title: SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target
    Article Snippet: .. Western blot assay and antibodiesProteins were resolved in 12% SDS-PAGE, transferred to PVDF membranes (GE), and incubated with primary antibodies against HA-Tag (C29F4) (3724, CST, USA), GAPDH (30201ES20, Yeasen, China), mCherry (ab125096, Abcam, UK), α-Tublin (11224-1-AP, Proteintech, USA), his (30404ES60, Yeasen, China). ..

    SDS Page:

    Article Title: SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target
    Article Snippet: .. Western blot assay and antibodiesProteins were resolved in 12% SDS-PAGE, transferred to PVDF membranes (GE), and incubated with primary antibodies against HA-Tag (C29F4) (3724, CST, USA), GAPDH (30201ES20, Yeasen, China), mCherry (ab125096, Abcam, UK), α-Tublin (11224-1-AP, Proteintech, USA), his (30404ES60, Yeasen, China). ..

    Incubation:

    Article Title: SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target
    Article Snippet: .. Western blot assay and antibodiesProteins were resolved in 12% SDS-PAGE, transferred to PVDF membranes (GE), and incubated with primary antibodies against HA-Tag (C29F4) (3724, CST, USA), GAPDH (30201ES20, Yeasen, China), mCherry (ab125096, Abcam, UK), α-Tublin (11224-1-AP, Proteintech, USA), his (30404ES60, Yeasen, China). ..

    Article Title: Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors
    Article Snippet: .. Cells were then incubated with the following antibodies: phospho DNA-PKcs S2056 (ab18192, Abcam, 1:200), RAD51 (ab1837, Abcam, 1:200), RPA70 (ab176467, Abcam, 1:200), or HA-tag (#3724, Cell Signalling, 1:100). ..

    Article Title: Epitranscriptomic editing of the RNA N6-methyladenosine modification by dCasRx conjugated methyltransferase and demethylase
    Article Snippet: .. Membranes were blocked with 5% non-fat powdered milk (Sangon Biotech, #A600669-0250) and incubated overnight at 4° cold room with anti-HA (C29F4) rabbit mAb (Cell Signaling Technology, #3724), GAPDH (Proteintech, #60004-1-Ig), YTHDF1 (Proteintech, #17479-1-AP), YTHDF2 (Proteintech, #24744-1-AP) and YTHDF3 (Proteintech, #25537-1-AP) antibodies in TBST (TBS + 0.5% Tween-20) with 1% bovine serum albumin (BSA, Sangon Biotech, # A600332-0100). ..

    Chromatin Immunoprecipitation:

    Article Title: ZHX2 Promotes HIF1α Oncogenic Signaling in Triple-Negative Breast Cancer
    Article Snippet: .. ChIP was performed with HA tag (Cell Signaling, 3724). ..

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    Cell Signaling Technology Inc rabbit anti α tubulin
    Consequences of PRDM12 mutations. ( a ) The hemagglutinin (HA)-tagged PRDM12 polyalanine expansion mutant showed lower expression levels in COS-7 cells than did wild-type PRDM12; expression was recovered by MG132. HA-PRDM12 signals were normalized to those of <t>α-tubulin</t> and GFP (transfection-efficiency control). The transfected mutant formed aggregates in the nucleus and cytoplasm in HEK-293T cells (bottom). Red, PRDM12 (fluorescent detection of the anti-HA tag); blue, nuclei (DAPI staining). ( b ) Wild-type Prdm12 induced robust dimethylation on H3K9 in Xenopus neurula, but CIP-associated missense mutants were functionless. Xenopus animal cap cells were microinjected with Myc- Prdm12 (wild type and mutant), Wnt8 and Chrd mRNA and cultured until mid-neurula stage (stage 15). H3K9me2 signals were normalized to Prdm12 (Myc) and total H3. ( c ) The p.His289Leu alteration impairs Prdm12-G9a interaction. Myc-Prdm12 and FLAG-G9a were expressed in COS-7 cells (Input) and immunoprecipitated using anti-Myc (IP: Myc). Bound G9a was normalized to amounts of Prdm12 in the immunoprecipitation (IP) fraction and G9a protein in the lysate. According to the structural model for the PRDM12 zinc finger domains, His289 (orange) is one of the residues (cyan) that coordinate the zinc ion. Prdm12ΔZF is an artificial mutant lacking zinc fingers. IgG, immunoglobulin G. ( d ) Mutation-altered residues Ile102 and Trp160 (orange) have hydrophobic interactions with other residues (cyan) in the core of the PRDM12-PR domain. Introduction of a polar side chain (p.Ile102Asn) or a disulfide-bond partner (p.Trp160Cys) into the hydrophobic core may affect the structure of the PR domain. The bar graphs in a – c represent mean values of n biological replicates, and error bars represent s.d. Statistical differences between control (wild type) and Prdm12 mutants are indicated. ns, not significant. * P
    Rabbit Anti α Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti α tubulin/product/Cell Signaling Technology Inc
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    99
    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
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    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
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    Consequences of PRDM12 mutations. ( a ) The hemagglutinin (HA)-tagged PRDM12 polyalanine expansion mutant showed lower expression levels in COS-7 cells than did wild-type PRDM12; expression was recovered by MG132. HA-PRDM12 signals were normalized to those of α-tubulin and GFP (transfection-efficiency control). The transfected mutant formed aggregates in the nucleus and cytoplasm in HEK-293T cells (bottom). Red, PRDM12 (fluorescent detection of the anti-HA tag); blue, nuclei (DAPI staining). ( b ) Wild-type Prdm12 induced robust dimethylation on H3K9 in Xenopus neurula, but CIP-associated missense mutants were functionless. Xenopus animal cap cells were microinjected with Myc- Prdm12 (wild type and mutant), Wnt8 and Chrd mRNA and cultured until mid-neurula stage (stage 15). H3K9me2 signals were normalized to Prdm12 (Myc) and total H3. ( c ) The p.His289Leu alteration impairs Prdm12-G9a interaction. Myc-Prdm12 and FLAG-G9a were expressed in COS-7 cells (Input) and immunoprecipitated using anti-Myc (IP: Myc). Bound G9a was normalized to amounts of Prdm12 in the immunoprecipitation (IP) fraction and G9a protein in the lysate. According to the structural model for the PRDM12 zinc finger domains, His289 (orange) is one of the residues (cyan) that coordinate the zinc ion. Prdm12ΔZF is an artificial mutant lacking zinc fingers. IgG, immunoglobulin G. ( d ) Mutation-altered residues Ile102 and Trp160 (orange) have hydrophobic interactions with other residues (cyan) in the core of the PRDM12-PR domain. Introduction of a polar side chain (p.Ile102Asn) or a disulfide-bond partner (p.Trp160Cys) into the hydrophobic core may affect the structure of the PR domain. The bar graphs in a – c represent mean values of n biological replicates, and error bars represent s.d. Statistical differences between control (wild type) and Prdm12 mutants are indicated. ns, not significant. * P

    Journal: Nature genetics

    Article Title: Transcriptional regulator PRDM12 is essential for human pain perception

    doi: 10.1038/ng.3308

    Figure Lengend Snippet: Consequences of PRDM12 mutations. ( a ) The hemagglutinin (HA)-tagged PRDM12 polyalanine expansion mutant showed lower expression levels in COS-7 cells than did wild-type PRDM12; expression was recovered by MG132. HA-PRDM12 signals were normalized to those of α-tubulin and GFP (transfection-efficiency control). The transfected mutant formed aggregates in the nucleus and cytoplasm in HEK-293T cells (bottom). Red, PRDM12 (fluorescent detection of the anti-HA tag); blue, nuclei (DAPI staining). ( b ) Wild-type Prdm12 induced robust dimethylation on H3K9 in Xenopus neurula, but CIP-associated missense mutants were functionless. Xenopus animal cap cells were microinjected with Myc- Prdm12 (wild type and mutant), Wnt8 and Chrd mRNA and cultured until mid-neurula stage (stage 15). H3K9me2 signals were normalized to Prdm12 (Myc) and total H3. ( c ) The p.His289Leu alteration impairs Prdm12-G9a interaction. Myc-Prdm12 and FLAG-G9a were expressed in COS-7 cells (Input) and immunoprecipitated using anti-Myc (IP: Myc). Bound G9a was normalized to amounts of Prdm12 in the immunoprecipitation (IP) fraction and G9a protein in the lysate. According to the structural model for the PRDM12 zinc finger domains, His289 (orange) is one of the residues (cyan) that coordinate the zinc ion. Prdm12ΔZF is an artificial mutant lacking zinc fingers. IgG, immunoglobulin G. ( d ) Mutation-altered residues Ile102 and Trp160 (orange) have hydrophobic interactions with other residues (cyan) in the core of the PRDM12-PR domain. Introduction of a polar side chain (p.Ile102Asn) or a disulfide-bond partner (p.Trp160Cys) into the hydrophobic core may affect the structure of the PR domain. The bar graphs in a – c represent mean values of n biological replicates, and error bars represent s.d. Statistical differences between control (wild type) and Prdm12 mutants are indicated. ns, not significant. * P

    Article Snippet: Proteins were detected using mouse anti-HA, rabbit anti–α-tubulin (Cell Signaling, 2144) and rabbit anti-acetylated histone 3 (Cell Signaling, 9677) followed by HRP-conjugated rabbit anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (Cell Signaling, 7074).

    Techniques: Mutagenesis, Expressing, Transfection, Staining, Cell Culture, Immunoprecipitation, Zinc-Fingers

    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

    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