tgf β2 neutralizing antibody  (R&D Systems)

Journal: Cyborg and Bionic Systems

Article Title: Bioinspired Nanocomposite for Targeted Immunoengineering and Improved Tendon Regeneration

doi: 10.34133/cbsystems.0503

Figure Lengend Snippet: KGN@PB@CM modulates macrophage polarization and restores immune homeostasis in vitro. (A) Representative immunofluorescence images showing iNOS (M1 marker, red) expression in RAW 264.7 macrophages under different treatment conditions. DAPI (blue) was used for nuclear staining. (B) Quantitative analysis of iNOS fluorescence intensity. (C) Representative dual-staining images for CD206 (M2 marker, green) and F4/80 (pan-macrophage marker, red). (D) Quantification of CD86 (M1 marker) fluorescence intensity. (E) Representative immunofluorescence images showing CD206 + F4/80 + macrophages in different groups. (F) Quantitative analysis of CD206 fluorescence intensity. (G to L) RT-qPCR analysis of M1-related genes ( iNOS , CD86 , TNF-α , and IL-6 ) and M2-related genes ( Arg-1 , CD206 , and IL-10 ) in different treatment groups. (M to Q) ELISA results showing the concentrations of inflammatory cytokines IL-1β, IL-6, TNF-α and anti-inflammatory cytokines IL-10 and TGF-β1 in macrophage supernatants. Data are presented as means ± SD, n = 5; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: IL-1β, IL-6, TNF-α, IL-10, and TGF-β1 levels were assessed by ELISA kits (MULTI SCIENCES, China) following the manufacturer’s instructions.

Techniques: In Vitro, Immunofluorescence, Marker, Expressing, Staining, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: Wild-type C57BL/6 mice (n=12/group) were injected intravitreally with either LV-TGF-β2 or LV-null vectors. ( A ) Weekly IOP measurements demonstrate significant and sustained IOP elevation in LV-TGF-β2-injected mice compared with LV-null controls. ( B ) Immunofluorescence staining of anterior segment cross-sections for α-SMA and MIF shows co-localization of MIF with the TM marker α-SMA, indicating increased MIF expression in the TM of LV-TGF-β2 injected mice. ( C-E ) qPCR ( C ) and western blot ( D, E ) analyses of anterior segment tissues show upregulation of MIF and CD74 accompanied by suppression of Blimp-1 in LV-TGF-β2-injected mice. Increased expression of profibrotic markers, including α-SMA, Fn-1, and MYOC, was also evident in LV-TGF-β2-injected mice. Western blot bands were quantified by densitometric analysis using ImageJ and normalized to the housekeeping control β-actin, and protein levels are presented as relative expression (AU) ( E ). Data represent mean ± SD. **p<0.005, ***p<0.0005, and ****p<0.0001. Two-way ANOVA with Tukey’s multiple-comparison test (A), unpaired t-test (C, E).

Article Snippet: Recombinant human TGF-β2 (302-B2-010/CF) protein was purchased from R&D Systems (Minneapolis, MN).

Techniques: Injection, Immunofluorescence, Staining, Marker, Expressing, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: Primary HTMCs (n=3) were challenged with TGF-β2 (10ng/mL), rMIF (100ng/mL), or pro-inflammatory cytokine pool (CP; TNF-α, IL-1β, IL-6, and IL-17 100ng/mL) for 24h. (A) qPCR analysis shows significant upregulation of MIF and CD74 transcripts accompanied by reduced Blimp-1 expression in response to glaucomatous stressors. ( B ) Immunofluorescence staining demonstrates increased MIF expression following TGF-β2 or CP treatment. (C) Western blot analysis confirms induction of MIF and CD74 with concurrent suppression of Blimp-1 at the protein level. Data represent mean ± SD. **p<0.005, ***p<0.0005, and ***p<0.0001, One-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: Recombinant human TGF-β2 (302-B2-010/CF) protein was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: Primary HTMCs (n=3) were challenged with rMIF (100 ng/mL) in the presence or absence of MIF inhibitor 4-IPP (100µM) or the metabolites Agm or Thia (100 ng/mL) for 24h. Cells treated with TGF-β2 (10ng/mL) served as a positive control. (A-B) Western blot analysis confirms activation of the RhoA/ROCK/pMLC signaling pathway in response to MIF treatment, which is significantly attenuated by MIF inhibition with 4-IPP, Agm, or Thia ( A ). Western blot bands were quantified by densitometric analysis using ImageJ and normalized to the housekeeping control β-actin, and protein levels are presented as relative expression (AU) ( B ). (C) Immunofluorescence analysis shows that MIF induces pMLC staining, comparable to that observed with TGF-β2 stimulation, which is significantly attenuated by MIF inhibition with 4-IPP, Agm, or Thia. Data represent mean ± SD. *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001, One-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: Recombinant human TGF-β2 (302-B2-010/CF) protein was purchased from R&D Systems (Minneapolis, MN).

Techniques: Positive Control, Western Blot, Activation Assay, Inhibition, Control, Expressing, Immunofluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: OHT was induced in WT C57BL/6 mice (n=12) by intravitreal injection of LV-TGF-β2. LV-null-injected mcie served as controls. Following OHT induction, extracellular vesicle-loaded agmatine (EV-Agm; 0.1 µg/eye) was applied topically once daily. (A) Weekly IOP measurements over 9 weeks show that EV-Agm treatment significantly reduces IOP compared with untreated LV-TGF-β2-injected mice. (B) qPCR analysis of anterior segment tissue demonstrates that EV-Agm significantly reduces expression of Mif, Cd74, pro-inflammatory cytokines ( Il-1 β , Il-6 ), and cytoskeletal/ECM markers (α -Sma, Myoc, Fn-1 ), while restoring Blimp-1 expression compared with untreated LV-TGF-β2-injected mice. *p<0.05, **p<0.005, ###,***p<0.0005, and ####,****p<0.0001. Two-way ANOVA with Tukey’s multiple comparison (A), one-way ANOVA with Tukey’s multiple comparison (B). In panel A, * denotes LV-null vs. LV-TGF-β2, and # denotes LV-TGF-β2 vs. LV-TGF-β2+EV-Agm comparisons.

Article Snippet: Recombinant human TGF-β2 (302-B2-010/CF) protein was purchased from R&D Systems (Minneapolis, MN).

Techniques: Injection, Expressing, Comparison

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: GO and KEGG pathway enrichment analyses of DEGs in MCF-7 cells after GGCT knockdown. (A) GO enrichment analysis for biological processes (up-regulated and down-regulated genes), and KEGG pathway analysis of DEGs, using samples collected 3 days after knockdown. The top 10 biological process terms ranked according to p-Values are shown. (B) Hypothetical schema of cell cycle regulation following GGCT knockdown in MCF-7 cells, constructed based on DEGs enriched in the KEGG pathway ‘hsa04110: Cell cycle’ (http://www.kegg.jp). (C) Relative mRNA expression patterns of GGCT, TGF-β2, CDKN1A (p21 Cip1 ), and CDKN2B (p15 INK4b ) were measured with qRT-PCR. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using two-tailed Student’s t-test for pairwise comparisons. CDKN1A, Cyclin dependent kinase inhibitor 1A; CDKN2B, cyclin dependent kinase inhibitor 2B; DEGs, differentially expressed genes; GGCT, γ-glutamylcyclotransferase; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; TGF-β2, transforming growth factor-β2.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Knockdown, Construct, Expressing, Quantitative RT-PCR, Two Tailed Test, Reverse Transcription, Polymerase Chain Reaction

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: TGF-β2/SMAD signaling axis regulates p15 INK4b and p21 Cip1 expression in GGCT-depleted MCF-7 cells. (A) mRNA expression levels of GGCT and TGF-β2 were analyzed by qRT-PCR 3 days after transfection with GGCT- and/or TGF-β2-siRNA. (B) Western blot analysis of GGCT and TGF-β2 expression 4 days after transfection with GGCT- and/or TGF-β2-siRNA. (C) Western blotting analysis of p15 INK4b , p21 Cip1 , phospho-SMAD2 (pSMAD2), SMAD2, phospho-SMAD3 (pSMAD3), SMAD3, GGCT, and α-tubulin in MCF-7 at 4 days after transfection with GGCT-siRNA and/or TGF-β2-siRNA, or non-target control siRNA. (D) Western blotting analysis of p15 INK4b , p21 Cip1 , pSMAD3, SMAD3, GGCT, and α-tubulin in MCF-7 cells at 4 days after transfection with GGCT-siRNA and/ or SMAD3-siRNA, or non-target control siRNA. (E) The number of viable cells in the trypan blue dye exclusion test and (F) representative images of MCF- 7 cells at 4 days after transfection with the indicated siRNAs. Scale bar: 200 μm; n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 vs. control, †p<0.05; ††p<0.01, and †††p<0.001 vs. GGCT, one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; pSMAD, phosphorylated SMAD; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: p15 INK4b , p21 Cip1 , and their upstream regulator TGF-β2 are involved in the induction of cellular senescence following GGCT-KD in MCF-7 cells. (A) Representative images of SA-β-Gal staining 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT, p15, and p21. Scale bar: 50 μm. (B) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. (C) Representative images of SA-β-Gal staining at 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT and TGF-β2. Scale bar: 50 μm. (D) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. n=3 per group; *p<0.05, **p<0.01, ***p<0.001, using one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; KD, knockdown; SA-β-Gal, senescence-associated β-galactosidase; siRNA, smallinterfering RNA; TGF-β2, transforming growth factor-β2.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Staining, Transfection, Knockdown