flag tag  (Proteintech)

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

Journal: eBioMedicine

Article Title: USP25 regulates atherosclerosis by restricting RIPK1-mediated inflammatory responses

doi: 10.1016/j.ebiom.2026.106213

Figure Lengend Snippet: USP25 interacts with RIPK1 through the USP domain. (A) Schematic diagram of the proteomic screening for USP25-interacting proteins. (B) Two-dimensional plot of USP25-binding proteins, with the Y axis showing protein intensity and the X axis showing protein molecular weight. (C) List of potential substrates with unique peptides ≥2 identified by the mass spectrometry analysis. (D–E) Whole cell lysates of BMDMs were immunoprecipitated with anti-USP25 (D) or anti-RIPK1 (E) antibodies, followed by Western blot analysis. Rabbit IgG served as negative control. (F–G) HEK293 cells were co-transfected with FLAG-USP25 and MYC-RIPK1 plasmids for 24 h. Whole-cell lysates were immunoprecipitated with anti-FLAG (F) or anti-MYC (G) antibodies, followed by Western blot analysis. (H) BMDMs were left untreated or treated with ox-LDL (50 μg/mL) for 30 min before lysis. Whole-cell lysates were immunoprecipitated with anti-USP25 antibody, followed by Western blot analysis. (I) Subcellular distribution of USP25 (red) and RIPK1 (green) in BMDMs treated with or without ox-LDL (50 μg/mL) for 30 min was detected by immunofluorescence. Scale bar: 5 μm. (J) Schematic diagram of structural domains and truncation mutants of USP25. (K) HEK293 cells were co-transfected with indicated plasmids for 24 h. Cell lysates were immunoprecipitated with anti-FLAG antibody and further analysed by Western blot with indicated antibodies.

Article Snippet: Antibodies against FLAG (Cat#: 20543-1-AP, 1: 1000, RRID: AB_11232216 ), MYC (Cat#: 60003-2-Ig, 1: 1000, RRID: AB_2734122 ), HA (Cat#: 51064-2-AP, 1: 1000, RRID: AB_11042321 ), CD36 (Cat#: 18836-1-AP, 1:1000, RRID: AB_10597244 ), ASK1 (Cat#: 28201-1-AP, 1: 1000, RRID: AB_2782957 ), MLKL (Cat#: 66675-1-Ig, 1:1000, RRID: AB_2882029 ), and p-ASK1 (Cat#: 28846-1-AP, 1: 1000, RRID: AB_2881220 ) were purchased from Proteintech (Wuhan, China).

Techniques: Binding Assay, Molecular Weight, Mass Spectrometry, Immunoprecipitation, Western Blot, Negative Control, Transfection, Lysis, Immunofluorescence