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mouse anti ha monoclonal antibody  (Proteintech)


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    Structured Review

    Proteintech mouse anti ha monoclonal antibody
    eIF3m interacted with FAdV-4 ORF1B protein in LMH cells. (A and B) eIF3m interacted with exogenous ORF1B. LMH cells co-transfected with 1 μg/well of p3 × flag-ORF1B and 1 μg/well pCAGGS-HA-eIF3m were lysed at 48 h, and cell lysates were immunoprecipitated with an anti-flag (A) or <t>anti-HA</t> (B) antibody followed by western blot with anti-HA and anti-flag antibodies. (C) The interaction of eIF3m with endogenous ORF1B. LMH cells transfected with 1 μg/well of pCAGGS-HA-eIF3m or empty vector were infected with CH/HNJZ/2015 at an MOI of 0.01 at 24 hpt. Cell lysates were immunoprecipitated with an anti-HA antibody followed by western blot with anti-HA and anti-ORF1B antibodies. (D) eIF3m and ORF1B were co-localized in cytoplasm. LMH cell co-transfected with 1 μg/well of pEGFP-ORF1B and 1 μg/well of pDsRed-eIF3m were fixed at 24 h. After stained with DAPI, the fluorescence was observed by using a confocal microscope.
    Mouse Anti Ha Monoclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1654 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+ha+monoclonal+antibody/HA+Tag+Antibody/pmc12887161-44-13-18
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    mouse anti ha monoclonal antibody - by Bioz Stars, 2026-09
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    Images

    1) Product Images from "eIF3m promotes fowl adenovirus serotype 4 replication via interacting with ORF1B protein"

    Article Title: eIF3m promotes fowl adenovirus serotype 4 replication via interacting with ORF1B protein

    Journal: Poultry Science

    doi: 10.1016/j.psj.2026.106566

    eIF3m interacted with FAdV-4 ORF1B protein in LMH cells. (A and B) eIF3m interacted with exogenous ORF1B. LMH cells co-transfected with 1 μg/well of p3 × flag-ORF1B and 1 μg/well pCAGGS-HA-eIF3m were lysed at 48 h, and cell lysates were immunoprecipitated with an anti-flag (A) or anti-HA (B) antibody followed by western blot with anti-HA and anti-flag antibodies. (C) The interaction of eIF3m with endogenous ORF1B. LMH cells transfected with 1 μg/well of pCAGGS-HA-eIF3m or empty vector were infected with CH/HNJZ/2015 at an MOI of 0.01 at 24 hpt. Cell lysates were immunoprecipitated with an anti-HA antibody followed by western blot with anti-HA and anti-ORF1B antibodies. (D) eIF3m and ORF1B were co-localized in cytoplasm. LMH cell co-transfected with 1 μg/well of pEGFP-ORF1B and 1 μg/well of pDsRed-eIF3m were fixed at 24 h. After stained with DAPI, the fluorescence was observed by using a confocal microscope.
    Figure Legend Snippet: eIF3m interacted with FAdV-4 ORF1B protein in LMH cells. (A and B) eIF3m interacted with exogenous ORF1B. LMH cells co-transfected with 1 μg/well of p3 × flag-ORF1B and 1 μg/well pCAGGS-HA-eIF3m were lysed at 48 h, and cell lysates were immunoprecipitated with an anti-flag (A) or anti-HA (B) antibody followed by western blot with anti-HA and anti-flag antibodies. (C) The interaction of eIF3m with endogenous ORF1B. LMH cells transfected with 1 μg/well of pCAGGS-HA-eIF3m or empty vector were infected with CH/HNJZ/2015 at an MOI of 0.01 at 24 hpt. Cell lysates were immunoprecipitated with an anti-HA antibody followed by western blot with anti-HA and anti-ORF1B antibodies. (D) eIF3m and ORF1B were co-localized in cytoplasm. LMH cell co-transfected with 1 μg/well of pEGFP-ORF1B and 1 μg/well of pDsRed-eIF3m were fixed at 24 h. After stained with DAPI, the fluorescence was observed by using a confocal microscope.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Infection, Staining, Fluorescence, Microscopy

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    Incubation:

    Article Title: TGEV nonstructural protein ORF3b upregulates the expression of SLA-DR at the transcriptional level in monocyte-derived porcine dendritic cells.
    Article Snippet: .. Next, the membranes were blocked with 5 % skim milk for 2 h at 37 ◦C and incubated with the following primary antibodies (1:1000) for 12 h at 4 ◦C: rabbit anti-SLA-DR monoclonal antibody (Novus Biologicals, USA); mouse anti-HA monoclonal antibody (Proteintech, China); mouse anti-β-actin monoclonal antibody (Proteintech, China); and an anti-TGEV-N protein monoclonal antibody provided by our laboratory. ..

    Article Title: Epitope mapping targeting the K205R protein of African swine fever virus using nanobody as a novel tool
    Article Snippet: Following three washes with PBST, the membranes were visualized using an ECL substrate (Beijing Solarbio Science & Technology Co., Ltd.). .. In a separate step, the PVDF membranes were blocked and subsequently incubated with primary antibodies for 2 h at RT, specifically, mouse anti-HA monoclonal antibody (mAb; 1:2,000; Proteintech Group, Inc.) and anti-α-tubulin (1:5,000; Proteintech Group, Inc.). .. After washing three times with PBST, HRP-conjugated goat anti-mouse IgG (1:5,000; Jackson ImmunoResearch Laboratories, Inc.) was incubated for 1 h at 37°C as the secondary antibody.

    Article Title: Epitope mapping targeting the K205R protein of African swine fever virus using nanobody as a novel tool.
    Article Snippet: Following three washes with PBST, the membranes were visualized using an ECL substrate (Beijing Solarbio Science & Technology Co., Ltd.). .. In a separate step, the PVDF membranes were blocked and subsequently incubated with primary antibodies for 2 h at RT, specifically, mouse anti-HA monoclonal antibody (mAb; 1:2,000; Proteintech Group, Inc.) and anti-α-tubulin (1:5,000; Proteintech Group, Inc.). .. After washing three times with PBST, HRP-conjugated goat anti-mouse IgG (1:5,000; Jackson ImmunoResearch Laboratories, Inc.) was incubated for 1 h at 37°C as the secondary antibody.

    Article Title: TGEV non-structural protein ORF3b up-regulates the expression of SLA-DR at transcriptional level in monocyte-derived porcine dendritic cells
    Article Snippet: .. Following that, membranes were sealed with 5% skim milk for 2h at 37°C and incubated with the appropriate primary antibodies (1:1000) below for overnight at 4 °C: rabbit anti-SLA-DR monoclonal antibody (Novus Biologicals, USA); mouse anti-HA monoclonal antibody (proteintech, China); mouse anti-β-actin monoclonal antibody (proteintech, China); an anti-TGEV-N protein monoclonal antibody which provided by our laboratory. ..

    Article Title: Adenosine deaminase promotes goose astrovirus genotype II replication in GEF cells
    Article Snippet: 48 h later, the cells were washed with chilled PBS and then lysed with IP lysis buffer (Beyotime, China) for 30 min on ice. .. After centrifuging at 10,000 rpm for 10 min at 4 °C, the supernatants were incubated with mouse anti-HA monoclonal antibody ( mAb ) (Proteintech, China) (dilution 1:2000) at 4 °C overnight, and then mixed with protein G-agarose beads for further 8 h at 4 °C. ..

    Sequencing:

    Article Title: Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor
    Article Snippet: .. The following antibodies were used: HL-1, a mouse monoclonal anti-the linker sequence between ligand binding repeat (LR) 4 and LR5 of LDLR antibody ( , ); a rabbit anti-MT1-MMP monoclonal antibody (Abcam, ab51074); a mouse anti-MT1-MMP monoclonal antibody (EMD Millipore, MAB3329); a rabbit anti-HA polyclonal antibody (ProteinTech, 51064-2-AP); a mouse anti-HA monoclonal antibody (ProteinTech, 66006-2-lg); a DylightTM 680-conjugated rabbit anti-HA antibody (Rockland, 600-444-384); a DylightTM 800-conjugated rabbit anti-HA antibody (Rockland, 600-445-384); a mouse anti-actin monoclonal antibody (ProteinTech, 66009-1-Ig); a mouse anti-Na + /K + -ATPase antibody (BD Biosciences, 610993); and a mouse anti-transferrin receptor monoclonal antibody (BD Biosciences, 612125). ..

    Ligand Binding Assay:

    Article Title: Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor
    Article Snippet: .. The following antibodies were used: HL-1, a mouse monoclonal anti-the linker sequence between ligand binding repeat (LR) 4 and LR5 of LDLR antibody ( , ); a rabbit anti-MT1-MMP monoclonal antibody (Abcam, ab51074); a mouse anti-MT1-MMP monoclonal antibody (EMD Millipore, MAB3329); a rabbit anti-HA polyclonal antibody (ProteinTech, 51064-2-AP); a mouse anti-HA monoclonal antibody (ProteinTech, 66006-2-lg); a DylightTM 680-conjugated rabbit anti-HA antibody (Rockland, 600-444-384); a DylightTM 800-conjugated rabbit anti-HA antibody (Rockland, 600-445-384); a mouse anti-actin monoclonal antibody (ProteinTech, 66009-1-Ig); a mouse anti-Na + /K + -ATPase antibody (BD Biosciences, 610993); and a mouse anti-transferrin receptor monoclonal antibody (BD Biosciences, 612125). ..



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    eIF3m interacted with FAdV-4 ORF1B protein in LMH cells. (A and B) eIF3m interacted with exogenous ORF1B. LMH cells co-transfected with 1 μg/well of p3 × flag-ORF1B and 1 μg/well pCAGGS-HA-eIF3m were lysed at 48 h, and cell lysates were immunoprecipitated with an anti-flag (A) or <t>anti-HA</t> (B) antibody followed by western blot with anti-HA and anti-flag antibodies. (C) The interaction of eIF3m with endogenous ORF1B. LMH cells transfected with 1 μg/well of pCAGGS-HA-eIF3m or empty vector were infected with CH/HNJZ/2015 at an MOI of 0.01 at 24 hpt. Cell lysates were immunoprecipitated with an anti-HA antibody followed by western blot with anti-HA and anti-ORF1B antibodies. (D) eIF3m and ORF1B were co-localized in cytoplasm. LMH cell co-transfected with 1 μg/well of pEGFP-ORF1B and 1 μg/well of pDsRed-eIF3m were fixed at 24 h. After stained with DAPI, the fluorescence was observed by using a confocal microscope.
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    eIF3m interacted with FAdV-4 ORF1B protein in LMH cells. (A and B) eIF3m interacted with exogenous ORF1B. LMH cells co-transfected with 1 μg/well of p3 × flag-ORF1B and 1 μg/well pCAGGS-HA-eIF3m were lysed at 48 h, and cell lysates were immunoprecipitated with an anti-flag (A) or <t>anti-HA</t> (B) antibody followed by western blot with anti-HA and anti-flag antibodies. (C) The interaction of eIF3m with endogenous ORF1B. LMH cells transfected with 1 μg/well of pCAGGS-HA-eIF3m or empty vector were infected with CH/HNJZ/2015 at an MOI of 0.01 at 24 hpt. Cell lysates were immunoprecipitated with an anti-HA antibody followed by western blot with anti-HA and anti-ORF1B antibodies. (D) eIF3m and ORF1B were co-localized in cytoplasm. LMH cell co-transfected with 1 μg/well of pEGFP-ORF1B and 1 μg/well of pDsRed-eIF3m were fixed at 24 h. After stained with DAPI, the fluorescence was observed by using a confocal microscope.
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    eIF3m interacted with FAdV-4 ORF1B protein in LMH cells. (A and B) eIF3m interacted with exogenous ORF1B. LMH cells co-transfected with 1 μg/well of p3 × flag-ORF1B and 1 μg/well pCAGGS-HA-eIF3m were lysed at 48 h, and cell lysates were immunoprecipitated with an anti-flag (A) or <t>anti-HA</t> (B) antibody followed by western blot with anti-HA and anti-flag antibodies. (C) The interaction of eIF3m with endogenous ORF1B. LMH cells transfected with 1 μg/well of pCAGGS-HA-eIF3m or empty vector were infected with CH/HNJZ/2015 at an MOI of 0.01 at 24 hpt. Cell lysates were immunoprecipitated with an anti-HA antibody followed by western blot with anti-HA and anti-ORF1B antibodies. (D) eIF3m and ORF1B were co-localized in cytoplasm. LMH cell co-transfected with 1 μg/well of pEGFP-ORF1B and 1 μg/well of pDsRed-eIF3m were fixed at 24 h. After stained with DAPI, the fluorescence was observed by using a confocal microscope.
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    ( A ) Western blot showing specificity of the XND-1 antibody. Western analysis was performed on protein extracts from wild-type N2 worms and two independent xnd-1 mutant strains. A distinct band corresponding to XND-1 (red square) is detected only in the N2 extract, confirming the specificity of the antibody. No signal is observed in the mutant strains, consistent with loss of xnd-1 expression (see also ). ( B ) 3xHA::HIM-17 and anti-XND-1 staining do not overlap with one another or with the DNA axes. Shown here are 3D renderings of confocal stacks from the mitotic zone, early-middle pachytene, and mid-late pachytene regions. ( C ) Localization of XND-1 is normal in him-17(ok424 M-Z- ) mutants (anti-XND-1, pink; DNA/DAPI, green) (top). Localization of 3xHA::HIM-17 is unaffected in xnd-1(ok709 ) mutants <t>(anti-HA,</t> pink; DNA/DAPI, green) (bottom).
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    ( A ) Western blot showing specificity of the XND-1 antibody. Western analysis was performed on protein extracts from wild-type N2 worms and two independent xnd-1 mutant strains. A distinct band corresponding to XND-1 (red square) is detected only in the N2 extract, confirming the specificity of the antibody. No signal is observed in the mutant strains, consistent with loss of xnd-1 expression (see also ). ( B ) 3xHA::HIM-17 and anti-XND-1 staining do not overlap with one another or with the DNA axes. Shown here are 3D renderings of confocal stacks from the mitotic zone, early-middle pachytene, and mid-late pachytene regions. ( C ) Localization of XND-1 is normal in him-17(ok424 M-Z- ) mutants (anti-XND-1, pink; DNA/DAPI, green) (top). Localization of 3xHA::HIM-17 is unaffected in xnd-1(ok709 ) mutants <t>(anti-HA,</t> pink; DNA/DAPI, green) (bottom).
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    Proteintech mouse monoclonal antibody anti ha
    ( A ) Western blot showing specificity of the XND-1 antibody. Western analysis was performed on protein extracts from wild-type N2 worms and two independent xnd-1 mutant strains. A distinct band corresponding to XND-1 (red square) is detected only in the N2 extract, confirming the specificity of the antibody. No signal is observed in the mutant strains, consistent with loss of xnd-1 expression (see also ). ( B ) 3xHA::HIM-17 and anti-XND-1 staining do not overlap with one another or with the DNA axes. Shown here are 3D renderings of confocal stacks from the mitotic zone, early-middle pachytene, and mid-late pachytene regions. ( C ) Localization of XND-1 is normal in him-17(ok424 M-Z- ) mutants (anti-XND-1, pink; DNA/DAPI, green) (top). Localization of 3xHA::HIM-17 is unaffected in xnd-1(ok709 ) mutants <t>(anti-HA,</t> pink; DNA/DAPI, green) (bottom).
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    Covance mouse monoclonal anti ha antibody
    DISC1–PML interaction is required for NPC proliferation in the developing cortex. A, lysates from human embryonic kidney (HEK) cells co-transfected with DISC1, PML and WT IE1 or IE1-L174P were immunoprecipitated with an anti-PML antibody and immunoblotted with an <t>anti-HA</t> antibody. B, lysates from HEK cells cotransfected with DISC1, IE1, and WT PML or mutant PML lacking the IE1-binding site (PMLΔIE1) were immunoprecipitated with an anti-PML antibody and immunoblotted with an anti-HA antibody. C, HEK cells cotransfected with PML and HA-tagged WT DISC1, or mutant DISC1 lacking the PML-binding site (DISC1ΔPML), were immunoprecipitated with the PML antibody and immunoblotted with the HA antibody. D, mouse embryos electroporated with various constructs at E13.5 were pulse labeled with BrdU (50 mg/kg) for 2 h at E15.5. Bar graph represents the percentage of GFP- and BrdU-double positive cells over total GFP-positive cells in the VZ/SVZ. Green , cells transfected with GFP, DISC1 RNAi, and DISC1 constructs; red , BrdU-positive cells; arrowheads indicate GFP- and BrdU-double positive cells. The scale bar represents 20 μm. Graph shows mean +/− SEM (GFP: n = 4, DISC1 RNAi: n = 6, DISC1 RNAi + Wt DISC1: n = 4, DISC1 RNAi + DISC1ΔPML: n = 3, Tukey’s multiple comparison test ∗ p < 0.05; one-way ANOVA: F(3,13) = 6.067, p = 0.0082). BrdU, bromodeoxyuridine; E13.5, embryonic day 13.5; E15.5, embryonic day 15.5; HA, hemagglutinin; NPC, neural progenitor cell; IE1, immediate early 1; SZ, subventricular zone; VZ, ventricular zone; PML, promyelocytic leukemia.
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    Abmart Inc anti ha tag mouse monoclonal antibody
    (A) Schematic diagram of RNF20 and its deletion mutants. (B) Luciferase reporter assay of HEK293T cells transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and RNF20 (wild-type or mutant) expression plasmids or control vector. (C, D) HEK293T cells were transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and Myc-RIG-I or Myc-MDA5, together with C-terminal truncation mutants of RNF20 (upper panels). Immunoblotting was performed with anti-Myc and anti-β-tubulin antibodies (lower panels). (E) HEK293T cells were co-transfected with Myc-MDA5 and RNF20-HA (wild-type or mutant) expression plasmids. Cell lysates were immunoprecipitated with <t>anti-HA</t> antibody and analyzed by immunoblotting with the indicated antibodies. (F) Co-immunoprecipitation analysis of the polyubiquitination of MDA5 in HEK293T cells transfected with Myc-MDA5, RNF20-Flag (wild-type or mutant), and HA-ubiquitin expression plasmids. (G) Quantification of MDA5 ubiquitination levels in immunoprecipitated samples. (H) Amino acid sequence alignment of the CARD domains of RIG-I and MDA5 showing the conserved “KENW” motif. (I, J) Co-immunoprecipitation analysis of the polyubiquitination of human RIG-I or MDA5 (wild-type or K-to-R mutants) in HEK293T cells transfected with Myc-RIG-I or Myc-MDA5, Flag-RNF20, and HA-ubiquitin expression plasmids. (K, L) Quantification of RIG-I and MDA5 ubiquitination levels in immunoprecipitated samples. (M-P) Immunoblot and quantification analysis of HEK293T cells transiently transfected with RNF20-HA and either RIG-I-Flag or RIG-I-dKENW-Flag, or MDA5-Flag or MDA5-dKENW-Flag, followed by VSV-GFP infection (MOI = 1.0) for 24h . Cell lysates were analyzed by immunoblotting with anti-Flag, anti-HA, and anti-β-tubulin antibodies. Data are shown as mean ± SD from three independent experiments (n = 3). ns: no significance, p < 0.05, * p < 0.01, ** p < 0.001.(Note: pcDNA3.1: empty vector control).
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    Image Search Results


    eIF3m interacted with FAdV-4 ORF1B protein in LMH cells. (A and B) eIF3m interacted with exogenous ORF1B. LMH cells co-transfected with 1 μg/well of p3 × flag-ORF1B and 1 μg/well pCAGGS-HA-eIF3m were lysed at 48 h, and cell lysates were immunoprecipitated with an anti-flag (A) or anti-HA (B) antibody followed by western blot with anti-HA and anti-flag antibodies. (C) The interaction of eIF3m with endogenous ORF1B. LMH cells transfected with 1 μg/well of pCAGGS-HA-eIF3m or empty vector were infected with CH/HNJZ/2015 at an MOI of 0.01 at 24 hpt. Cell lysates were immunoprecipitated with an anti-HA antibody followed by western blot with anti-HA and anti-ORF1B antibodies. (D) eIF3m and ORF1B were co-localized in cytoplasm. LMH cell co-transfected with 1 μg/well of pEGFP-ORF1B and 1 μg/well of pDsRed-eIF3m were fixed at 24 h. After stained with DAPI, the fluorescence was observed by using a confocal microscope.

    Journal: Poultry Science

    Article Title: eIF3m promotes fowl adenovirus serotype 4 replication via interacting with ORF1B protein

    doi: 10.1016/j.psj.2026.106566

    Figure Lengend Snippet: eIF3m interacted with FAdV-4 ORF1B protein in LMH cells. (A and B) eIF3m interacted with exogenous ORF1B. LMH cells co-transfected with 1 μg/well of p3 × flag-ORF1B and 1 μg/well pCAGGS-HA-eIF3m were lysed at 48 h, and cell lysates were immunoprecipitated with an anti-flag (A) or anti-HA (B) antibody followed by western blot with anti-HA and anti-flag antibodies. (C) The interaction of eIF3m with endogenous ORF1B. LMH cells transfected with 1 μg/well of pCAGGS-HA-eIF3m or empty vector were infected with CH/HNJZ/2015 at an MOI of 0.01 at 24 hpt. Cell lysates were immunoprecipitated with an anti-HA antibody followed by western blot with anti-HA and anti-ORF1B antibodies. (D) eIF3m and ORF1B were co-localized in cytoplasm. LMH cell co-transfected with 1 μg/well of pEGFP-ORF1B and 1 μg/well of pDsRed-eIF3m were fixed at 24 h. After stained with DAPI, the fluorescence was observed by using a confocal microscope.

    Article Snippet: Commercial antibodies used in this study included rabbit anti-flag monoclonal antibody (240568AA1, proteintech), mouse anti-HA monoclonal antibody (66006-2-Ig, proteintech), rabbit anti-GAPDH monoclonal antibody (A19056, ABclonal).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Infection, Staining, Fluorescence, Microscopy

    ( A ) Western blot showing specificity of the XND-1 antibody. Western analysis was performed on protein extracts from wild-type N2 worms and two independent xnd-1 mutant strains. A distinct band corresponding to XND-1 (red square) is detected only in the N2 extract, confirming the specificity of the antibody. No signal is observed in the mutant strains, consistent with loss of xnd-1 expression (see also ). ( B ) 3xHA::HIM-17 and anti-XND-1 staining do not overlap with one another or with the DNA axes. Shown here are 3D renderings of confocal stacks from the mitotic zone, early-middle pachytene, and mid-late pachytene regions. ( C ) Localization of XND-1 is normal in him-17(ok424 M-Z- ) mutants (anti-XND-1, pink; DNA/DAPI, green) (top). Localization of 3xHA::HIM-17 is unaffected in xnd-1(ok709 ) mutants (anti-HA, pink; DNA/DAPI, green) (bottom).

    Journal: eLife

    Article Title: Genetic and physical interactions reveal overlapping and distinct contributions to meiotic double-strand break formation in C. elegans

    doi: 10.7554/eLife.96458

    Figure Lengend Snippet: ( A ) Western blot showing specificity of the XND-1 antibody. Western analysis was performed on protein extracts from wild-type N2 worms and two independent xnd-1 mutant strains. A distinct band corresponding to XND-1 (red square) is detected only in the N2 extract, confirming the specificity of the antibody. No signal is observed in the mutant strains, consistent with loss of xnd-1 expression (see also ). ( B ) 3xHA::HIM-17 and anti-XND-1 staining do not overlap with one another or with the DNA axes. Shown here are 3D renderings of confocal stacks from the mitotic zone, early-middle pachytene, and mid-late pachytene regions. ( C ) Localization of XND-1 is normal in him-17(ok424 M-Z- ) mutants (anti-XND-1, pink; DNA/DAPI, green) (top). Localization of 3xHA::HIM-17 is unaffected in xnd-1(ok709 ) mutants (anti-HA, pink; DNA/DAPI, green) (bottom).

    Article Snippet: Mouse monoclonal anti-HA (Cell Signaling), chicken polyclonal anti-GFP (AbCam), mouse monoclonal anti-FLAG HRP-conjugated (Sigma), and polyclonal anti-XND-1 ( ) antibodies were diluted in blocking solution at 1:1000, 1:5000, 1:2000, and 1:2500, respectively, and left to incubate overnight at 4°C.

    Techniques: Western Blot, Mutagenesis, Expressing, Staining

    ( A ) Immunofluorescence analysis of HIM-5::HA in dsb-1 mutants. DAPI (blue) stains DNA, anti-HA marks endogenously tagged HIM-5 (green), and pHTP-3 S285 (red) labels chromosome axes. HIM-5 appears localized in nuclei in pre-meiotic stages (indicated by arrows). However, after the transition zone (TZ), HIM-5 loses its nuclear localization. ( B ) Immunofluorescence analysis of HIM-5::HA in him-5::3XHA control . Top: DAPI (blue) stains DNA, anti-HA marks endogenously tagged HIM-5 (green), and pHTP-3 S285 (red) labels chromosome axes. Bottom: Zoomed-in region where HIM-5 (yellow) can be observed localizing in pre-TZ nuclei through mid-pachytene. ( C ) Top: C. elegans gonad fixed and stained with DAPI to show the organization and distribution of the nuclei along the Prophase I. Bottom: Live imaging of nuclei in the transition zone (leptotene–zygotene) and middle-pachytene. eaIs15 (Ppie-1::him-5::GFP) is visualized in freshly dissected gonads by GFP fluorescence (green), and DNA by DRAQ5 (red). In dsb-1 mutants, HIM-5 is nuclear in the transition zone and then only appears in cytoplasmic puncta by middle pachytene.

    Journal: eLife

    Article Title: Genetic and physical interactions reveal overlapping and distinct contributions to meiotic double-strand break formation in C. elegans

    doi: 10.7554/eLife.96458

    Figure Lengend Snippet: ( A ) Immunofluorescence analysis of HIM-5::HA in dsb-1 mutants. DAPI (blue) stains DNA, anti-HA marks endogenously tagged HIM-5 (green), and pHTP-3 S285 (red) labels chromosome axes. HIM-5 appears localized in nuclei in pre-meiotic stages (indicated by arrows). However, after the transition zone (TZ), HIM-5 loses its nuclear localization. ( B ) Immunofluorescence analysis of HIM-5::HA in him-5::3XHA control . Top: DAPI (blue) stains DNA, anti-HA marks endogenously tagged HIM-5 (green), and pHTP-3 S285 (red) labels chromosome axes. Bottom: Zoomed-in region where HIM-5 (yellow) can be observed localizing in pre-TZ nuclei through mid-pachytene. ( C ) Top: C. elegans gonad fixed and stained with DAPI to show the organization and distribution of the nuclei along the Prophase I. Bottom: Live imaging of nuclei in the transition zone (leptotene–zygotene) and middle-pachytene. eaIs15 (Ppie-1::him-5::GFP) is visualized in freshly dissected gonads by GFP fluorescence (green), and DNA by DRAQ5 (red). In dsb-1 mutants, HIM-5 is nuclear in the transition zone and then only appears in cytoplasmic puncta by middle pachytene.

    Article Snippet: Mouse monoclonal anti-HA (Cell Signaling), chicken polyclonal anti-GFP (AbCam), mouse monoclonal anti-FLAG HRP-conjugated (Sigma), and polyclonal anti-XND-1 ( ) antibodies were diluted in blocking solution at 1:1000, 1:5000, 1:2000, and 1:2500, respectively, and left to incubate overnight at 4°C.

    Techniques: Immunofluorescence, Control, Staining, Imaging, Fluorescence

    DISC1–PML interaction is required for NPC proliferation in the developing cortex. A, lysates from human embryonic kidney (HEK) cells co-transfected with DISC1, PML and WT IE1 or IE1-L174P were immunoprecipitated with an anti-PML antibody and immunoblotted with an anti-HA antibody. B, lysates from HEK cells cotransfected with DISC1, IE1, and WT PML or mutant PML lacking the IE1-binding site (PMLΔIE1) were immunoprecipitated with an anti-PML antibody and immunoblotted with an anti-HA antibody. C, HEK cells cotransfected with PML and HA-tagged WT DISC1, or mutant DISC1 lacking the PML-binding site (DISC1ΔPML), were immunoprecipitated with the PML antibody and immunoblotted with the HA antibody. D, mouse embryos electroporated with various constructs at E13.5 were pulse labeled with BrdU (50 mg/kg) for 2 h at E15.5. Bar graph represents the percentage of GFP- and BrdU-double positive cells over total GFP-positive cells in the VZ/SVZ. Green , cells transfected with GFP, DISC1 RNAi, and DISC1 constructs; red , BrdU-positive cells; arrowheads indicate GFP- and BrdU-double positive cells. The scale bar represents 20 μm. Graph shows mean +/− SEM (GFP: n = 4, DISC1 RNAi: n = 6, DISC1 RNAi + Wt DISC1: n = 4, DISC1 RNAi + DISC1ΔPML: n = 3, Tukey’s multiple comparison test ∗ p < 0.05; one-way ANOVA: F(3,13) = 6.067, p = 0.0082). BrdU, bromodeoxyuridine; E13.5, embryonic day 13.5; E15.5, embryonic day 15.5; HA, hemagglutinin; NPC, neural progenitor cell; IE1, immediate early 1; SZ, subventricular zone; VZ, ventricular zone; PML, promyelocytic leukemia.

    Journal: The Journal of Biological Chemistry

    Article Title: Cytomegalovirus-encoded immediate early 1 protein perturbs neural progenitor proliferation via interfering with host PML–DISC1 interaction

    doi: 10.1016/j.jbc.2026.111269

    Figure Lengend Snippet: DISC1–PML interaction is required for NPC proliferation in the developing cortex. A, lysates from human embryonic kidney (HEK) cells co-transfected with DISC1, PML and WT IE1 or IE1-L174P were immunoprecipitated with an anti-PML antibody and immunoblotted with an anti-HA antibody. B, lysates from HEK cells cotransfected with DISC1, IE1, and WT PML or mutant PML lacking the IE1-binding site (PMLΔIE1) were immunoprecipitated with an anti-PML antibody and immunoblotted with an anti-HA antibody. C, HEK cells cotransfected with PML and HA-tagged WT DISC1, or mutant DISC1 lacking the PML-binding site (DISC1ΔPML), were immunoprecipitated with the PML antibody and immunoblotted with the HA antibody. D, mouse embryos electroporated with various constructs at E13.5 were pulse labeled with BrdU (50 mg/kg) for 2 h at E15.5. Bar graph represents the percentage of GFP- and BrdU-double positive cells over total GFP-positive cells in the VZ/SVZ. Green , cells transfected with GFP, DISC1 RNAi, and DISC1 constructs; red , BrdU-positive cells; arrowheads indicate GFP- and BrdU-double positive cells. The scale bar represents 20 μm. Graph shows mean +/− SEM (GFP: n = 4, DISC1 RNAi: n = 6, DISC1 RNAi + Wt DISC1: n = 4, DISC1 RNAi + DISC1ΔPML: n = 3, Tukey’s multiple comparison test ∗ p < 0.05; one-way ANOVA: F(3,13) = 6.067, p = 0.0082). BrdU, bromodeoxyuridine; E13.5, embryonic day 13.5; E15.5, embryonic day 15.5; HA, hemagglutinin; NPC, neural progenitor cell; IE1, immediate early 1; SZ, subventricular zone; VZ, ventricular zone; PML, promyelocytic leukemia.

    Article Snippet: For immunofluorescence and immunoblotting, HA-tagged proteins were detected with a rat monoclonal anti-HA antibody (ROCHE) or a mouse monoclonal anti-HA antibody (Covance).

    Techniques: Transfection, Immunoprecipitation, Mutagenesis, Binding Assay, Construct, Labeling, Comparison

    (A) Schematic diagram of RNF20 and its deletion mutants. (B) Luciferase reporter assay of HEK293T cells transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and RNF20 (wild-type or mutant) expression plasmids or control vector. (C, D) HEK293T cells were transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and Myc-RIG-I or Myc-MDA5, together with C-terminal truncation mutants of RNF20 (upper panels). Immunoblotting was performed with anti-Myc and anti-β-tubulin antibodies (lower panels). (E) HEK293T cells were co-transfected with Myc-MDA5 and RNF20-HA (wild-type or mutant) expression plasmids. Cell lysates were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting with the indicated antibodies. (F) Co-immunoprecipitation analysis of the polyubiquitination of MDA5 in HEK293T cells transfected with Myc-MDA5, RNF20-Flag (wild-type or mutant), and HA-ubiquitin expression plasmids. (G) Quantification of MDA5 ubiquitination levels in immunoprecipitated samples. (H) Amino acid sequence alignment of the CARD domains of RIG-I and MDA5 showing the conserved “KENW” motif. (I, J) Co-immunoprecipitation analysis of the polyubiquitination of human RIG-I or MDA5 (wild-type or K-to-R mutants) in HEK293T cells transfected with Myc-RIG-I or Myc-MDA5, Flag-RNF20, and HA-ubiquitin expression plasmids. (K, L) Quantification of RIG-I and MDA5 ubiquitination levels in immunoprecipitated samples. (M-P) Immunoblot and quantification analysis of HEK293T cells transiently transfected with RNF20-HA and either RIG-I-Flag or RIG-I-dKENW-Flag, or MDA5-Flag or MDA5-dKENW-Flag, followed by VSV-GFP infection (MOI = 1.0) for 24h . Cell lysates were analyzed by immunoblotting with anti-Flag, anti-HA, and anti-β-tubulin antibodies. Data are shown as mean ± SD from three independent experiments (n = 3). ns: no significance, p < 0.05, * p < 0.01, ** p < 0.001.(Note: pcDNA3.1: empty vector control).

    Journal: PLOS Pathogens

    Article Title: RNF20 dynamically regulates RIG-I and MDA5 transcription and degradation via nucleocytoplasmic translocation to balance antiviral signaling

    doi: 10.1371/journal.ppat.1013890

    Figure Lengend Snippet: (A) Schematic diagram of RNF20 and its deletion mutants. (B) Luciferase reporter assay of HEK293T cells transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and RNF20 (wild-type or mutant) expression plasmids or control vector. (C, D) HEK293T cells were transiently transfected with IFN-β-luc (120 ng), pRL-TK (60 ng), and Myc-RIG-I or Myc-MDA5, together with C-terminal truncation mutants of RNF20 (upper panels). Immunoblotting was performed with anti-Myc and anti-β-tubulin antibodies (lower panels). (E) HEK293T cells were co-transfected with Myc-MDA5 and RNF20-HA (wild-type or mutant) expression plasmids. Cell lysates were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting with the indicated antibodies. (F) Co-immunoprecipitation analysis of the polyubiquitination of MDA5 in HEK293T cells transfected with Myc-MDA5, RNF20-Flag (wild-type or mutant), and HA-ubiquitin expression plasmids. (G) Quantification of MDA5 ubiquitination levels in immunoprecipitated samples. (H) Amino acid sequence alignment of the CARD domains of RIG-I and MDA5 showing the conserved “KENW” motif. (I, J) Co-immunoprecipitation analysis of the polyubiquitination of human RIG-I or MDA5 (wild-type or K-to-R mutants) in HEK293T cells transfected with Myc-RIG-I or Myc-MDA5, Flag-RNF20, and HA-ubiquitin expression plasmids. (K, L) Quantification of RIG-I and MDA5 ubiquitination levels in immunoprecipitated samples. (M-P) Immunoblot and quantification analysis of HEK293T cells transiently transfected with RNF20-HA and either RIG-I-Flag or RIG-I-dKENW-Flag, or MDA5-Flag or MDA5-dKENW-Flag, followed by VSV-GFP infection (MOI = 1.0) for 24h . Cell lysates were analyzed by immunoblotting with anti-Flag, anti-HA, and anti-β-tubulin antibodies. Data are shown as mean ± SD from three independent experiments (n = 3). ns: no significance, p < 0.05, * p < 0.01, ** p < 0.001.(Note: pcDNA3.1: empty vector control).

    Article Snippet: The antibodies IgG were purchased from the indicated manufacturer’s, including HRP-conjugated anti-mouse or rabbit IgG (Cell Signaling Technologies, 7076 and 7074), Alexa Fluor 488-conjugated Goat anti-rabbit IgG (Sangon Biotech, D110061), Cy3-conjugated Goat anti-mouse IgG (Sangon Biotech, D110088), Anti-Flag tag mouse monoclonal antibody (Sangon Biotech, D191041), Anti-HA tag mouse monoclonal antibody (Abmart, 26D11), Anti-Myc tag mouse monoclonal antibody (Abmart, 19C2), Anti-GFP tag mouse monoclonal antibody (ABclonal,AE012), anti-β-tubulin (Abmart, 2H4), anti-Lamin B1 (Beyotime, AF1408), anti-RNF20 (proteintech, 21625–1-AP), anti-RNF20 (Santa Cruz biotechnology, sc-517358), anti-RIG-I (Cell Signaling Technologies, 3743), anti-MDA5 (Cell Signaling Technologies, 5321), anti-MAVS (Cell Signaling Technologies, 3993), anti-TBK1 (Cell Signaling Technologies, 3504), anti- p -TBK1 (Cell Signaling Technologies, 5483), anti-IRF3 (Cell Signaling Technologies, 11904), anti- p -IRF3 (Cell Signaling Technologies, 4947), anti-STING (Cell Signaling Technologies, 13647S), anti-STAT1 (Sangon Biotech, D120084).

    Techniques: Luciferase, Reporter Assay, Transfection, Mutagenesis, Expressing, Control, Plasmid Preparation, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Sequencing, Infection