flag tag  (Proteintech)

Journal: Materials Today Bio

Article Title: Inhalable engineered probiotic outer membrane vesicles co-expressing multiple mpox antigens induce potent specific systemic and mucosal immune responses

doi: 10.1016/j.mtbio.2026.103089

Figure Lengend Snippet: Design, Preparation, and Characterization of ABM@OMV. ( A ) Schematic illustrating the preparation of ABM@OMV. The pThioHisA plasmid, encoding Trx-A9R and ClyA-B6R-M1R fusion proteins, was transformed into Δ lpxM Escherichia coli Nissle 1917 (ΔEcN). OMVs were harvested via ultracentrifugation. ( B ) Bacterial lysates analyzed by SDS-PAGE and stained with Coomassie blue. Lanes: Marker; uninduced ΔEcN; ΔEcN + IPTG; uninduced BMΔEcN; BMΔEcN + IPTG; uninduced ABMΔEcN; ABMΔEcN + IPTG. Black boxes indicate ClyA-B6R-M1R and Trx-A9R fusion proteins. ( C ) Western blot analysis of proteins of interest expressed in ABMΔECN bacteria. Anti-His tag antibody detected Trx-A9R; anti-Flag tag antibody detected ClyA-B6R-M1R. Lane order is the same as in B . ( D ) Dynamic light scattering (DLS) size distribution profiles of Δ lpxM OMVs, BM@OMV, and ABM@OMV. ( E ) Zeta potential measurements of Δ lpxM OMVs, BM@OMV, and ABM@OMV ( n = 3). ( F ) Transmission electron microscopy (TEM) images of Δ lpxM OMVs, BM@OMV, and ABM@OMV. Scale bar: 100 nm. ( G ) Western blot analysis of proteins of interest expressed in ABM@OMV. Lanes: Δ lpxM OMVs, BM@OMV, ABM@OMV. ( H ) Endotoxin levels in Δ lpxM EcN-derived OMV and wild-type EcN-derived OMV, as measured by Limulus amebocyte lysate (LAL) assay ( n = 3). ( I ) Representative western blot showing the stability of proteins of interest in Δ lpxM OMVs, BM@OMV, and ABM@OMV following storage at 4 °C (left) or −80 °C (right) for the indicated time periods for the indicated time periods. ( J, K ) Changes in particle size ( J ) and zeta potential ( K ) of OMVs stored at 4 °C and −80 °C ( n = 3). All data were analyzed with GraphPad Prism 8 and are presented as mean ± SD. For panel H , statistical significance between two groups was determined by an unpaired two-tailed t -test. ∗∗∗ P < 0.001.

Article Snippet: The membrane was blocked with 5% BSA (BSA0020, Biosharp, China), then incubated with anti-His tag antibody (1:10,000, HY-P809476X, Med ChemExpress, USA) and anti-FLAG antibody (1:10,000, HY- P80111 , Med ChemExpress, USA), respectively for A9R and B6R-M1R, followed by HRP-conjugated secondary antibodies.

Techniques: Plasmid Preparation, Transformation Assay, SDS Page, Staining, Marker, Western Blot, Bacteria, FLAG-tag, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Derivative Assay, LAL Assay, Two Tailed Test

Journal: Transboundary and Emerging Diseases

Article Title: Toxoplasma gondii KCR is a Noncanonical Modulator of CSF2 Signaling that Targets the CSF2Rα–JAK2/STAT5 Axis

doi: 10.1155/tbed/8426765

Figure Lengend Snippet: Protein interactions and eukaryotic protein acquisition. (A) Schematic overview of the screening strategy and identification of T. gondii KCR. (B) Co‐immunoprecipitation identification of the interaction between KCR and murine CSF2Rα input: cell lysates from HEK 293T cells co‐transfected with pcDNA3.1‐KCR and pCAGGS‐CSF2R for 24 h; IP: KCR, CSF2α or IgG: immunoprecipitation was performed using Flag‐tag mouse mAb, His‐tag mouse mAb or mouse IgG; IB: KCR or CSF2α: immunoblot analysis was performed using Flag‐tag rabbit mAb or His‐tag rabbit pAb. (C) Acquisition of KCR eukaryotic protein. Lane M: standard molecular marker for protein; lane 1: cell lysates from HEK 293T cells transfected with pcDNA3.1‐KCR for 24 h; lane 2: purified KCR eukaryotic protein. (D) Western blot analysis of KCR M: standard molecular marker for protein; lane 3: his‐tag in purified KCR was identified by His‐tag mouse mAb.

Article Snippet: Flag‐tag mouse monoclonal antibody (mAb) (#M20008), His‐tag mouse mAb (# M20001 ), and mouse IgG (#B30010M) were purchased from Abmart Biotech, Inc. (Shanghai, China).

Techniques: Immunoprecipitation, Transfection, FLAG-tag, Western Blot, Marker, Purification

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 promotes m 3 C modification of Tcf7 mRNA and its genome-specific loops of Tox in CD8 + T cells. (A) Venn plot illustrates the overlap of downregulated genes from RNA-seq, m 3 C-seq, and Mettl8-binding genes from RIP-seq. (B) Mettl8 occupancy at the Tcf7 gene loci is revealed through m 3 C-seq (WT and Mettl8 −/− ) of EG7-OVA tumor-infiltrating OT-I cells and RIP-seq (Mettl8-tdTomato-Flag) of B16F10 tumor-infiltrating CD44 + CD8 + T cells. The binding peaks on Tcf7 loci are depicted. The m 3 C tracks are all plotted on a consistent scale. (C) The RNA decay assay demonstrates the remaining Tcf7 mRNA of CD8 + T cells from the spleens of WT and Mettl8 −/− mice detected by qRT-PCR, normalized to t = 0. (D) Heatmaps display changes in total Tcf1-targeting genes between WT and Mettl8 −/− EG7-OVA tumor-infiltrating OT-I cells and Mettl8-targeting genes in B16F10 tumor-infiltrating CD44 + CD8 + T cells of Mettl8-tdTomato-Flag mice as detected by CUT&Tag. (E) Diamond graphs exhibit chromatin interactions in WT and Mettl8 −/− tumor-infiltrating OT-I cells at the Tox gene loci (top), with CUT&Tag and ATAC-seq tracks, and gene structures on the bottom. An enlarged view highlights the signal profiles across the Tox gene region. (F) co-IP of Tcf1 by anti-Flag magnetic beads in CD3 + T cells from the spleens of Mettl8-tdTomato-Flag (RPT) and WT mice. IB, immunoblot. (G) co-IP of Tcf1 by Flag-tagged Mettl8 protein with anti-Flag magnetic beads after co-transfection into HEK293T cells. (H) Single-cell transcription levels of representative genes illustrated in the UMAP plot. Transcription levels are color coded: gray, not expressed; blue, expressed. (I) Schematic diagram of the tumor model: Mettl8 fl/fl Cd4 cre mice were subcutaneously injected with 2 × 10 5 B16F10 cells and harvested after 13 days. (J) Representative flow cytometry plots and cumulative data show the frequency of Tcf1 + Tox + cells gated on tumor-infiltrating CD8 + CD44 + T cells (right). n = 6 per group. (K) Schematic diagram of the OT-I–transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. Representative flow cytometry plots and cumulative data show the frequency of Tox + cells gated on Tcf1 + OT-I cells. n = 6 per group. (L) The MFI of Tox gated on Tcf1 + OT-I cells of the mice in K. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: .

Article Snippet: In briefly, cells were sorted enriched by ConA-magnetic beads and resuspended in wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCI, 0.5 mM spermidine; 1× protease inhibitor cocktail; 0.05% digitonin) and then incubated overnight with anti-Tcf1 (1:50, C63D9, cat. no. 2203; Cell Signaling Technology), anti-H3K27ac (1:50, cat. no. ab4729; Abcam), or anti-Flag (1:50, D6W5B, cat. no. 14793; Cell Signaling Technology).

Techniques: Modification, RNA Sequencing, Binding Assay, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Cotransfection, Single Cell, Injection, Flow Cytometry, Two Tailed Test