Journal: Frontiers in Immunology

Article Title: A fully human afucosylated anti-GARP antibody PBA-0111 drives anti-tumor immunity through combined ADCC induction and TGF-β1 pathway blockade

doi: 10.3389/fimmu.2026.1921265

Figure Lengend Snippet: Binding properties of PBA-0111 to GARP and GARP/TGF-β1. (A) Increasing concentrations of PBA-0111 were titrated on HEK293T cells overexpressing GARP, GARP/TGF-β1 or empty vector (EV) control, and the binding potency was analyzed by flow cytometry. (B) Increasing concentrations of PBA-0111, an ABBV-151-like antibody, a DS-1055a-like antibody, or an afucosylated isotype control were titrated on L-428 lymphoma cell line, which endogenously expresses GARP/TGF-β1 and the binding potency was analyzed by flow cytometry. Data in (A) and (B) are shown as mean ± standard deviation (SD) and are representative of at least three independent experiments performed in duplicates. (C) Flow cytometry analysis of binding of PBA-0111, DS-1055a-like, or ABBV-151-like antibody to human platelets at 10 µg/mL. Data are expressed as fold change in median fluorescence intensity (MdFI) relative to the afucosylated isotype control, (n=6 independent donors, **p=0.0062, n.s, p=0.06 comparing to PBA-0111, Kruskal-Wallis test). (D) Biolayer interferometry (BLI) binding analysis of PBA-0111 against recombinant extracellular GARP, GARP/TGF-β1, latency associated peptide (LAP), TGF-β1, latent TGF-β1, latent TGF-β2, latent TGF-β3, or GARP paralog leucine-rich repeat-containing protein 15 (LRRC15) at 500 nM. All samples were tested in at least three independent experiments in duplicate. (E) HEK-Blue™ TGF-β reporter assay. Raji GARP/TGF-β1 cells were pre-incubated with the indicated antibodies for 30 minutes and then co-cultured with HEK-Blue™ TGF-β cells. After 48h at 37 °C, SEAP activity was assessed using QUANTI-Blue™ Solution and optical density (OD) measured at 630 nm. Data shown as mean ± SD, n= 9, from 3 independent experiments. Statistical differences were assessed by Kruskal-Wallis test with Dunn´s multiple comparisons versus vehicle or afucosylated isotype control, **p<0.001. (F) Flow cytometry analysis of GARP/TGF-β1 complex formation on Raji-GARP cells. Cells were preincubated with 0.2 ng/mL to 50 µg/mL of PBA-0111, ABBV-151-like antibody, DS-1055a-like antibody, or afucosylated isotype control prior to addition of recombinant latent TGF-β1 at final concentration of 1.5 µg/mL. Complex formation was detected using an anti-LAP antibody. The graph is representative of at least three independent experiments. (G) TGF-β1 signalling was assessed in a Jurkat cell line overexpressing β8-GARP, stimulated with plate-bound anti-CD3 beads and soluble anti-CD28 in the presence of indicated antibodies (20 μg/mL) for 24 h at 37 °C. Cell lysates were analyzed by western blot and densitometric analysis of pSMAD2 bands was performed. (H) pSMAD2 band intensities from WB were normalized to the loading control (GAPDH) and then to isotype control,(n= 6 gels, ABBV-151 n=8 samples, 4 MHG-8 and 4 LGH.10.6). Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s multiple comparisons to IgG1.aF control, ***p=0.0002 (ABBV-151, n=8), **p=0.0037 (ADWA11). Statistical significance of PBA-0111 in single comparison to IgG1. aF was performed with Mann-Whitney analysis, p=0.002.

Article Snippet: Protein lysates were run in 4-20% Mini-Protean TGX Gel (BioRad), transferred to membranes followed by overnight incubation with anti-pSMAD2 antibody (Cell signalling technology, 3108L) and subsequent anti-rabbit IgG-HRP (R&D Systems, AF008) for one hour at RT.

Techniques: Binding Assay, Plasmid Preparation, Control, Flow Cytometry, Standard Deviation, Analysis, Fluorescence, Recombinant, Reporter Assay, Incubation, Cell Culture, Activity Assay, Concentration Assay, Western Blot, Comparison, MANN-WHITNEY

Journal: Frontiers in Neurology

Article Title: Transcriptomics-based analysis of muscle injury mechanism mediated by inflammatory imbalance in the Duchenne muscular dystrophy muscle microenvironment

doi: 10.3389/fneur.2026.1905030

Figure Lengend Snippet: PTPRC participates in TGF-β/SMAD signaling response representative WB bands for p-SMAD2 (Ser465/467), total-SMAD2 (t-SMAD2), and PTPRC across four experimental groups (si-NC / si-PTPRC ± TGF-β1), with GAPDH as the internal reference.

Article Snippet: Membranes were blocked with 5% BSA in TBST buffer (500 mM NaCl, 20 mM Tris–HCl, 0.2% Tween) for 1 h, then incubated overnight at 4 °C with primary antibodies against Myh6 (ab185967, Abcam, UK), MyoG (ab124800, Abcam, UK), PTPRC (ab281586, Abcam, UK), COL1A1 (ab260043, Abcam, UK), MMP9 (ab228402, Abcam, UK), and CTGF (ab6992, Abcam, UK), Phospho-SMAD2 (Ser465/467)(#3,108, CST, USA), SMAD2/3 (#3102, CST, USA).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Integrative transcriptomic and experimental analysis identifies GREM1 as a pro-metastatic mediator in lung adenocarcinoma

doi: 10.1186/s12967-026-08793-9

Figure Lengend Snippet: GREM1 interacts with BMP2 and regulates TGF-β/SMAD signaling pathway. ( A ) schematic diagram illustrates protein-protein interaction networks involving hub genes and EMT, glycolysis and TGF-β signaling pathways genes, based on findings from the STRING database. ( B ) 3D Binding model analysis(GREM1 in pink and BMP2 in green). ( C ) Immunofluorescence double staining detecting the colocalization and expression changes of GREM1 (red) and BMP2 (green) in the control group, GREM1 knockdown group (sh-GREM1-1), and overexpression group (oe-GREM1). ( D ) Co-immunoprecipitation assay verifying the direct binding between GREM1 and BMP2. ( E ) Western blot analysis of TGF-β/SMAD pathway components, including SMAD2/3, p-SMAD2, p-SMAD3, TGF-β1, BMP2, Smad1, Smad5 and Smad8. ( F ) IHC staining of GREM1 in tumor and adjacent normal tissues. ( G ) Representative images of GREM1 expression in immunohistochemical staining. ( H ) Kaplan–Meier curves for overall survival by GREM1 expression

Article Snippet: The protein was separated, transferred, and then incubated with 5% milk for 2 h. Antibodies including GREM1 (Santa Cruz, sc-515877), TGF-β1 (Santa Cruz, sc-130348), smad23 (Santa Cruz, sc-398844), p-smad2 (ABclonal, AP1007), p-smad3 (ABclonal, AP0727), smad1 (ABclonal, A19113), smad5 (ABclonal, A22749), smad8 (Santa Cruz, sc-518051), BMP2(ABclonal, A27101 ), snail(ABclonal, A11794), E-cadherin (Proteintech, Cat No. 20874-1-AP), N-cadherin (proteintech, Cat No. 22018-1-AP), Vimentin (Santa Cruz, sc-373717), HK2 (Proteintech, Cat No. 22029-1-AP), LDHA (ABclonal, A21893), PKM2 (ABclonal, A0268), TPI1(ABclonal, A2579) and β-Actin (ABclonal, AC038) were incubated with the protein overnight at 4 °C.

Techniques: Protein-Protein interactions, Binding Assay, Immunofluorescence, Double Staining, Expressing, Control, Knockdown, Over Expression, Co-Immunoprecipitation Assay, Western Blot, Immunohistochemistry, Immunohistochemical staining, Staining