mouse monoclonal anti ha  (Santa Cruz Biotechnology)

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: Journal of Virology

Article Title: AGPAT1 is a novel Chikungunya virus receptor on human cells

doi: 10.1128/jvi.01733-25

Figure Lengend Snippet: CHIKV uptake in Huh7 cells is related to the AGPAT1 protein levels. ( A ) Huh7 cells were transfected with siRNA to AGPAT1 (siAGPAT1) or a control non-targeting siRNA (siNT) and incubated for 48 h at 37°C. Western blotting showed a ~60% reduction in the AGPAT1 levels in the siAGPAT1-treated cells. ( B ) The siRNA-treated cells were incubated 48 h post-transfection (pt) with CHIKV (MOI 1) for 1 h on ice for the virus uptake assay. The relative CHIKV RNA levels are shown (left panel), where the CHIKV RNA level in the siNT-transfected cells at 1h post-infection (pi) was taken as 1. The viral titers determined by plaque assay are shown in the right panel. The data from four biological replicates and two technical replicates are shown. ( C ) Huh7 cells were transfected with the pAGPAT1 plasmid expressing the HA-tagged AGPAT1 or the empty vector pcDNA5. The cells were harvested at 24 and 48 h later. The cell lysates were western blotted with monoclonal anti-HA antibody to detect the expression of the HA-tagged AGPAT1. GAPDH was used as the loading control. ( D ) Huh7 cells were transfected with pAGPAT1-HA or pcDNA5, and 48 h later, they were incubated with CHIKV (MOI 5) for the virus binding assay. The relative CHIKV RNA levels are presented (left panel), where the CHIKV RNA level at 1 h pi was taken as 1. The right panel has the virus titers. The data from three biological replicates and two technical replicates are shown. ** P < 0.01, *** P < 0.001.

Article Snippet: The cells were stained for 2 h at room temperature with rabbit polyclonal anti-AGPAT1 antibody (ab235328; Abcam) for AGPAT1, mouse monoclonal anti-E1 (GTX135187; R&D systems) for CHIKV E1, goat polyclonal anti-EEA1 (ab206860; Abcam) for EEA1, rabbit monoclonal anti-HA (3724; Cell Signaling Tech) or mouse monoclonal anti-HA (2367; Cell Signaling Tech) for AGPAT1-HA, rabbit monoclonal anti-FLAG (14793; Cell Signaling Tech), or mouse monoclonal anti-FLAG (91878; Invitrogen) for E1-FLAG.

Techniques: Transfection, Control, Incubation, Western Blot, Virus, Infection, Plaque Assay, Plasmid Preparation, Expressing, Binding Assay

Journal: Journal of Virology

Article Title: AGPAT1 is a novel Chikungunya virus receptor on human cells

doi: 10.1128/jvi.01733-25

Figure Lengend Snippet: CHIKV binding and uptake in Huh7 cells ectopically expressing the AGPAT1 mutant proteins. ( A ) Huh7 cells were transfected with the plasmids expressing AGPAT1-HA or the mutant proteins, or the empty vector pcDNA5. The non-permeabilized cells were blocked and then stained with the rabbit monoclonal anti-HA antibody, followed by fixing and staining with anti-rabbit Alexa Fluor-568 antibody. The cells were imaged to examine the expression of the ectopically produced AGPAT1-HA or its mutants on the plasma membrane. ( B ) Huh7 cells were transfected with the plasmids expressing AGPAT1-HA or the mutant proteins, or the empty vector pcDNA5. The cells were washed at 48 h pt and incubated with CHIKV (MOI 50) on ice for 1 h and stained with CHIKV anti-E2 antibody to study the virus binding by microscopy. ( C ) The MIFD (25 ROI) data of the transfected Huh7 cells stained as above for the CHIKV binding are presented from three biological replicates. ( D ) Huh7 cells were transfected with the plasmids expressing AGPAT1-HA or the mutant proteins, or the empty vector pcDNA5. The cells were incubated 48 h later with CHIKV (MOI 3) for the virus uptake assay. The relative CHIKV RNA levels are presented, where the CHIKV RNA level in cells transfected with pcDNA5 was taken as 1. The data from five biological replicates and two technical replicates are shown. ( E ) Huh7 cells were transfected with the plasmids expressing AGPAT1-HA or the mutant proteins, or the empty vector pcDNA5. The cells were infected 48 h later with CHIKV (MOI 3) and incubated at 37°C. The virus titers in the culture supernatant at 6 h pi are presented. The data from four biological replicates and two technical replicates are shown. The statistical analysis on data shown as mean±SD was done by the one-way ANOVA with Dunnett’s multiple comparisons test, considering the pcDNA5-transfected cells as the control. *** P < 0.001, or ns = not significant.

Article Snippet: The cells were stained for 2 h at room temperature with rabbit polyclonal anti-AGPAT1 antibody (ab235328; Abcam) for AGPAT1, mouse monoclonal anti-E1 (GTX135187; R&D systems) for CHIKV E1, goat polyclonal anti-EEA1 (ab206860; Abcam) for EEA1, rabbit monoclonal anti-HA (3724; Cell Signaling Tech) or mouse monoclonal anti-HA (2367; Cell Signaling Tech) for AGPAT1-HA, rabbit monoclonal anti-FLAG (14793; Cell Signaling Tech), or mouse monoclonal anti-FLAG (91878; Invitrogen) for E1-FLAG.

Techniques: Binding Assay, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Staining, Produced, Clinical Proteomics, Membrane, Incubation, Virus, Microscopy, Infection, 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