mouse anti ha monoclonal antibody  (Proteintech)

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

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

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

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

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