Journal: Biotechnology Reports

Article Title: Comparative analysis of anti-MICA scFv affinities: Insights from three label-free biophysical methods and biological validation

doi: 10.1016/j.btre.2026.e00955

Figure Lengend Snippet: Characterization of Recombinant Proteins: MICA and anti-MICA scFvs. (A) Molecular model, shown as a ribbon representation, of the variable fragment of the anti-MICA scFvs. The framework is displayed in white, the light chain CDRs are shown in cyan, and the heavy chain CDRs are shown in yellow. The residues with mutations are shown as magenta spheres [residues 32 (CDR L1), 164 (CDR H1), and 188/190 (CDR H2)]. (B) Schematic diagram of the scFv gene. The modified pET-15b vector was used for the expression of the WT and Beta mutant scFvs, each carrying four mutations: I32Y in CDR1 of the VL, and S164F, P188W, and G190W in CDR1, CDR2, and CDR2 of the VH, respectively. Recombinant proteins were expressed in E. coli BL21(DE3). (C) SDS-PAGE analysis showing the purity of recombinant proteins: WT scFv, Beta mutant scFv, and MICA. Proteins were resolved on a 12% acrylamide gel under reducing conditions. SDS-PAGE results show the soluble fraction (SF), unbound protein (UBP), elution of purified scFv (E), renatured proteins (R) and inclusion bodies (IB). MW, molecular weight. (D-E) Western blot analysis confirming the identity of scFvs and MICA using an anti-HisTag antibody. For the identification of the WT and Beta mutant scFvs, Anti-6xHis Epitope Tag mouse monoclonal antibody conjugated with peroxidase (200-303-382) was used at a dilution of 1:1000. For the identification of MICA, a biotinylated Anti-MICA antibody (BAMO3 (BAFI300, BamOmaB)) and Streptavidin were used at a dilution of 1:2000. A total of 2 μg of purified protein was loaded. The negative control (Ctrl -) for MICA detection was WT scFv and MICA protein was used for scFv detection. Original gel is presented in Fig. S1, Supplementary information.

Article Snippet: The identity of MICA and scFvs proteins was confirmed by western blot using a HRP-conjugated anti-His tag monoclonal antibody (200-303-382, Rockland, USA).

Techniques: Recombinant, Modification, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Acrylamide Gel Assay, Purification, Molecular Weight, Western Blot, Negative Control

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