flag  (Cell Signaling Technology Inc)

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: Nature Communications

Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis

doi: 10.1038/s41467-026-71333-y

Figure Lengend Snippet: a Immuno-TLC of extracted gangliosides from CHO-Δ Slc33a1 (Ex1-5 del) cells transfected with a plasmid encoding human V5-tagged ST8SIA1, alone or in combination with a plasmid encoding the indicated N-terminally Flag-tagged SLC33A1 variants. Mock transfected cells were used as negative control. Total gangliosides were extracted and separated by TLC followed by staining with anti-9- O -Ac-GD3 mAb M-T6004 or anti-GD3 mAb R24. Representative plates from one of three independent experiments are shown. b Flow cytometric analysis of CHO-Δ Slc33a1 (Ex1-5 del) cells transiently transfected as described in ( a ) and stained with anti-9- O -Ac-GD3 mAb M-T6004. Positive cells were gated as shown in Supplementary Fig. . Data are presented as mean ± s.d. of n = 4 independent experiments. One-way ANOVA followed by Bonferroni’s post-hoc test. **** p < 0.0001 indicates a significant difference in comparison to ST8SIA1/SLC33A1-WT expressing cells. Western blot analysis was performed to validate the expression of all Flag-tagged SLC33A1 variants. Protein bands representing full-length SLC33A1 and the C-terminally truncated variant p.Y366* are marked by an arrow. The bands marked by asterisks are already present in the lysate of mock- and ST8SIA1 -transfected cells and represent non-specific cross-reactivity of the anti-Flag antibody with endogenous cellular proteins. Actin was used as loading controls. c Cartoon representation of the cryo-EM structure of SLC33A1 (PDB ID 9M0S) . The N- and C-terminal six-helix bundles of SLC33A1 are colored in green and beige, respectively. Side chains of the residues in position 110 and 113 are shown as sticks. Dotted red lines visualize the course of the helix axes of TMH2, TMH11, and TMH12. Detail enlargements of the SLC33A1 structure highlighting the positioning of A110 ( d ) and G509 ( e ). Helices are shown as semi-transparent ribbons with selected residues shown in stick representation. Hydrogen bonds are indicated as dotted lines. For clarity, only the main chain atoms of L474 and V471 are depicted. f Schematic representation of the membrane topology of SLC33A1. Positions of patient-derived SLC33A1 mutations are indicated. Source data and exact p values are provided as a file.

Article Snippet: Membranes were blocked in 3% BSA in PBS and incubated with rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.02 μg mL −1 ; Cell Signaling cat. 2368) overnight at 4 °C.

Techniques: Transfection, Plasmid Preparation, Negative Control, Staining, Comparison, Expressing, Western Blot, Variant Assay, Cryo-EM Sample Prep, Membrane, Derivative Assay