mouse mabs against flag  (Developmental Studies Hybridoma Bank)

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Receptor binding and fusogenicity of S proteins of omicron variants (A) Amino Acid sequence alignment of RBDs from different omicron variants. Dashed lines represent residues identical to WT. Residues of RBM are shown in red. (B and C) Cleavage of S proteins from different omicron variants. Plasmids encoding codon-optimized S proteins were transfected into HEK293T cells and the cells were then lysed at 40 h post-transfection. The expression of S proteins was detected by western blot using rabbit polyclonal anti-S2 antibodies. Actin served as the loading control. Experiments were done three times and one representative was shown. Band densities were quantified using ImageLab software to determine the cleaved/FL ratio and normalized to WT. FL S, full-length spike protein. (D) Binding of soluble hACE2 to S proteins of omicron variants. HEK293T cells transiently expressing the indicated S proteins were incubated with biotinylated hACE2 followed by Alexa Fluor 488-conjugated streptavidin and then analyzed by flow cytometry. Experiments were done three times, and one representative was shown. (E and F) Cell-cell fusion mediated by S proteins of omicron variants. HEK293T cells were co-transfected with indicated S protein and eGFP. The cells were detached with trypsin at 48h post-transfection and overlaid on the HEK293 cells expressing hACE2 or Calu3 cells. After 2 h of incubation, five randomly selected images of syncytia were captured using a fluorescent microscope. Fusion areas were quantified using ImageJ software and normalized to WT as fusion efficiency. Experiments were done three times in triplicate and one representative is shown. The statistical difference relative to WT was determined by one-way ANOVA with Dunnett’s multiple testing correction ( n = 3 for (C) and (D); n = 5 for (E) and (F)). The statistical difference between BA.2.86 and JN.1 was determined by a two-tailed t-test ( n = 3 for (C) and (D); n = 5 for (E) and (F)). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Scale bar = 200μm. See also .

Article Snippet: Mouse polyclonal against FLAG-tag antibodies , SinoBiological Inc (Beijing, China) , Cat#109143-MM13.

Techniques: Binding Assay, Sequencing, Transfection, Expressing, Western Blot, Control, Software, Incubation, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Transduction of various omicron S pseudovirions on 293/hACE2 and Calu3 cells (A) S proteins incorporation in pseudovirions. Pseudovirions with different omicron S proteins were pelleted down by centrifugation through 20% sucrose cushion and separated in a 10% SDS-PAGE. Detection of S proteins in pseudovirions was performed by Western blot using rabbit polyclonal anti-S2 antibodies. The p24 served as the loading controls. Experiments were done three times and one representative was shown. (B and C) Entry of pseudovirions of omicron variants. HEK 293/hACE2 and Calu3 cells were transduced with omicron S pseudovirions and lysed at 40 h post-transduction. The transduction efficiencies were determined according to luciferase activities. Experiments were done three times in triplicate and one representative is shown. The statistical difference relative to WT was determined by one-way ANOVA with Dunnett’s multiple testing correction ( n = 3). The statistical difference between BA.2.86 and JN.1 was determined by a two-tailed t-test ( n = 3). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse polyclonal against FLAG-tag antibodies , SinoBiological Inc (Beijing, China) , Cat#109143-MM13.

Techniques: Transduction, Centrifugation, SDS Page, Western Blot, Luciferase, Two Tailed Test

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Receptor binding of omicron variants to various animal ACE2s (A) Relative receptor binding efficiencies of RBDs of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, and JN.1 variants to various animal ACE2s. HEK293T transiently expressing different animal ACE2s were detached with EDTA and incubated with mouse Fc-tagged RBDs of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, or JN.1, followed by FITC-conjugated polyclonal goat anti-mouse antibodies. The cells were analyzed by flow cytometry. Receptor binding efficiencies were calculated according to WT/hACE2, set as 100%. Experiments were performed three times, and the averages are shown in the heatmap. (B) Statistic analyses of levels of receptor binding between different omicron variant S protein by Wilcoxon test. Significant differences indicated by p < 0.05 are highlighted in green background.

Article Snippet: Mouse polyclonal against FLAG-tag antibodies , SinoBiological Inc (Beijing, China) , Cat#109143-MM13.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry, Variant Assay

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Effect of L455S mutation on thermal and proteolytic stability on XBB.1.16 and BA.2.86 (A and B) Thermal stability of JN.1 compared to its parental BA.2.86 (left panel) and XBB.1.16–455S to XBB.1.16 (right panel). Pseudovirus of XBB.1.16, XBB.1.16–455S, BA.2.86 and JN.1 were centrifuged through 20% sucrose to remove serum and resuspended in serum-free DMEM, followed by incubation either at 37°C for the indicated time (A) or at the indicated temperature for 2 h (B). The virus suspension was used to transduce 293/hACE2 cells to access their remaining transduction capability. Experiments were performed three times in triplicate, and the representative one was shown. The statistical difference was determined by a multiple t-test ( n = 3). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (C) Proteolytic stability of JN.1 compared to its parental BA.2.86 (left panel) and XBB.1.16–455S to XBB.1.16 (right panel). Pseudovirus of XBB.1.16, XBB.1.16–455S, BA.2.86 and JN.1 were resuspended in serum-free DMEM and incubated with soluble hACE2(20 μg) at 37°C for 30 min, followed by incubation with TPCK-treated trypsin. The mixture was immediately boiled with loading buffer containing DTT and separated in a 10% SDS-PAGE. Detection was carried out using rabbit polyclonal anti-SARS-CoV-2 S2 antibodies (1:3000). The numbers below S protein blot are the relative quantification of ratio of S2’/S2 using Image Lab (Bio-Rad). Experiments were performed at least three times, and the representative one was shown.

Article Snippet: Mouse polyclonal against FLAG-tag antibodies , SinoBiological Inc (Beijing, China) , Cat#109143-MM13.

Techniques: Mutagenesis, Incubation, Virus, Suspension, Transduction, SDS Page, Quantitative Proteomics

Journal: Veterinary research

Article Title: The African swine fever virus B125R protein antagonizes JAK-STAT signalling by promoting the degradation of IFNAR2.

doi: 10.1186/s13567-025-01523-x

Figure Lengend Snippet: Figure 6 Proteomic analysis of the pB125R-host interactome. A HEK293T cells were transfected with Flag-pB125R (5 µg) for 24 h, lysed for co-IP with anti-Flag MAb, and subjected to western blot analysis with the indicated antibodies. High-throughput proteomic analysis was performed on immunoprecipitated samples via mass spectrometry (MS), with two replicates of each group. B and C, KEGG pathway analysis and GO category functional enrichment were performed for the 20 identified proteins. D The protein‒protein interaction network of proteins interacting with pB125R was analysed via the STRING database.

Article Snippet: Mouse Mab against Flag (66008-4-Ig) and MYC (60003-2-Ig) were obtained from Proteintech.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, High Throughput Screening Assay, Immunoprecipitation, Mass Spectrometry, Functional Assay

Journal: Biomolecules

Article Title: Pharmacological Inhibition of Astrocytic Transglutaminase 2 Facilitates the Expression of a Neurosupportive Astrocyte Reactive Phenotype in Association with Increased Histone Acetylation

doi: 10.3390/biom14121594

Figure Lengend Snippet: Irreversible inhibition of TG2 with the drug VA4 reduces interaction between TG2 and Zbtb7a. ( a ) Input controls of V5-TG2 and FLAG-Zbtb7a transfected into HEK 293TN cells treated with VA4 or vehicle control (DMSO). ( b ) Immunoprecipitation of V5-TG2 pulls down less FLAG-Zbtb7a in cell lysates that were treated with VA4. In ( a , b ) the position at which molecular weight markers (kDa) migrated is indicated at the left of the immunoblots. ( c ) Quantification of the amount of FLAG-Zbtb7a pulled down normalized to the amount of V5-TG2 immunoprecipitated. Treatment of cells with VA4 resulted in a significant reduction in the Zbtb7a co-immunoprecipitated with TG2 compared to DMSO control. Shown as mean and SEM (n = 5 samples per condition from 4 independent biological replicates, unpaired t -test *** p < 0.001). Original images can be found in .

Article Snippet: After blocking, primary antibodies against FLAG tag (CST 8146S), V5 tag (CST 13202S), GAPDH (Proteintech, Rosemont, IL, USA, 60004-1-Ig), acetylated histone H3 K9 (CST 9649S), or beta tubulin (rabbit polyclonal antibody, Proteintech 10094-1-AP) were added to the blots in fresh blocking buffer and incubated at 4 °C overnight.

Techniques: Inhibition, Transfection, Control, Immunoprecipitation, Molecular Weight, Western Blot