Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions in the presence or absence of FasL-LZ (25 ng/ml). Top: Representative flow cytometry histograms showing pS6(S240/44) staining in live CD4 + cells. Bottom: Fold induction of pS6(S240/44) in Th2-polarized (−/+ FasL-LZ) live CD4 + T cells. Fold induction was calculated by normalizing MFIs to NC WT cells. n = 4–5 for each group, from four to five independent experiments. (B and C) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions. n = 6 for each group, from six independent experiments. (B) Frequencies of IFNγ + Th2-polarized cells. (C) Th2-polarized cells were gated as FasL low , FasL mid , and FasL high . Frequencies of IFNγ + cells were measured in the indicated groups. (D–G) Fas was tagged with BioID2 on its intracellular C terminus (Fas-BioID) and stably expressed in Fas-deficient Jurkat cells. Fas-BioID Jurkat cells were cultured in the presence or absence of recombinant multimeric FasL (FasL-LZ). Jurkat cells expressing BioID alone were used as a control. (D) Venn diagram showing proteins identified following mass spectrometry analysis of biotinylated proteins (streptavidin pull-down) that were common between or specific to BioID-alone Jurkat cells versus Fas-BioID + FasL-LZ (P < 0.05, n = 3 for all groups). (E) Heatmap (row z-score) showing % normalized spectral abundance of proteins specifically upregulated in Fas-BioID ± FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells. (F) Pathway enrichment of Reactome gene sets was performed using Enrichr , with significantly enriched gene sets colored in blue; proteins specifically upregulated in Fas-BioID + FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells were used as input for pathway enrichment. (G) Normalized spectral abundance (%) of the indicated proteins in BioID-alone, Fas-BioID, and Fas-BioID + FasL-LZ Jurkat cells, measured by mass spectrometry. Statistical comparisons were made using ratio paired t tests (G). *P < 0.05, **P < 0.01. NC, negative control.

Article Snippet: Biotinylated proteins were pulled down using streptavidin agarose beads (Pierce) at 4°C overnight.

Techniques: Activation Assay, Flow Cytometry, Staining, Stable Transfection, Cell Culture, Recombinant, Expressing, Control, Mass Spectrometry, Negative Control

Journal: STAR Protocols

Article Title: Protocol to identify mechanosensitive nuclear proteins using tunable actomyosin contractility and proximity biotinylation in mammalian cells

doi: 10.1016/j.xpro.2025.104288

Figure Lengend Snippet: Inducible EGF-CA-RhoA and TurboID mediated biotinylation in HEK293-tet-RhoA-TurboID (A) EGFP-CA-RhoA expression in HEK293-tet-RhoA-TurboID can be controlled by tetracycline in a dose dependent (upper panel) and time dependent (lower panel) manner. γ-adaptin is used as loading control. (B) TurboID-catalyzed protein biotinylation in the presence of 50 μM Biotin for indicated time points. The biotinylated proteins were detected by streptavidin blotting. HA is used as loading control.

Article Snippet: Pierce Streptavidin Agarose , Thermo Fisher Scientific , #20349.

Techniques: Expressing, Control

Journal: STAR Protocols

Article Title: Protocol to identify mechanosensitive nuclear proteins using tunable actomyosin contractility and proximity biotinylation in mammalian cells

doi: 10.1016/j.xpro.2025.104288

Figure Lengend Snippet: Enrichment and biotinylation of nuclear proteins in HEK293-tet-RhoA-TurboID (A) HEK293-tet-RhoA-TurboID were treated with tetracycline for 2 h followed by biotin for 20 min. The stably expressing TurboID was detected by western blotting with anti-HA antibody. The biotinylated proteins in samples from cell lysate and nuclear fraction (streptavidin pulldown) were analyzed by western blotting with Alexa Fluor™ 680 Streptavidin Conjugate. (B) Western blot of indicated proteins in total cell lysate and nuclear fraction (streptavidin pulldown). Nucleolin and β-tubulin were used as nuclear and cytosolic marker respectively. (C) Quantification shows the normalized YAP expression from B. Replicates=4. Values are means ± s.d. ∗∗∗∗ p < 0.0001.

Article Snippet: Pierce Streptavidin Agarose , Thermo Fisher Scientific , #20349.

Techniques: Stable Transfection, Expressing, Western Blot, Marker