human fibronectin fn1 elisa kit  (Boster Bio)

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: Establishment and characterization of GC (Gemcitabine and Cisplatin)-resistant bladder cancer cell lines and identification of resistance-related proteins. ( A ) GC-resistant T24-R and UC3-R cell lines were generated by gradually increasing GC concentrations. Created in BioRender ( https://BioRender.com ). ( B ) Dose-response curves and calculated half-maximal inhibitory concentration (IC50) values for cisplatin (upper panel) and gemcitabine (lower panel) in parental (T24, UC3) and GC-resistant (T24-R, UC3-R) cell lines. Data are presented as the mean ± SD from at least three independent experiments. ( C ) Apoptosis rates of parental and resistant cell lines after treatment with cisplatin, as determined by flow cytometry. Data are presented as the mean ± SD ( n ≥ 3). ( D ) Transcriptomic and proteomic analyses identified FN1 (Fibronectin), EEF1A2 (Eukaryotic Translation Elongation Factor 1 Alpha 2), MRC2 (Mannose Receptor C-Type 2), RAB6B (RAB6B, Member RAS Oncogene Family), THBS1 (Thrombospondin 1), DYSF (Dysferlin), TMOD1 (Tropomodulin 1), NES (Nestin), and APOE (Apolipoprotein E) as overexpressed in GC-resistant cells. ( E ) RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) and Western blot confirmed FN1 overexpression in T24-R and UC3-R. ( F ) FN1 staining was stronger in GC-resistant bladder cancer tissues (Chemotherapy Sensitive Group: n = 6; Chemotherapy Resistant Group: n = 6). For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, ** p < 0.01

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Generated, Concentration Assay, Flow Cytometry, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Staining

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: ITGB4 is critical for FN1-mediated chemotherapy resistance in bladder cancer cells. ( A ) Differential expression analysis revealed that ITGB4 (highlighted by the red box) was significantly overexpressed in T24-R cells compared to T24, suggesting a role in FN1-mediated resistance. ( B ) Structural modeling shows multiple binding sites between FN1 (blue ribbon) and ITGB4 (green ribbon). ( C ) The interaction between FN1 and ITGB4 had a binding score of −318.75 with a confidence score of 96%, indicating a stable interaction. ( D ) The Co-IP experiment confirmed that there is a mutual binding interaction between FN1 and ITGB4. ( E ) Adding rFN1 to resistant strains increased FAK (Y397) phosphorylation and inhibited apoptosis, whereas ITGB4 silencing reversed these effects, highlighting the dependency of FN1-mediated resistance on ITGB4 expression and activation. For panels ( A , C , E ), a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Quantitative Proteomics, Binding Assay, Co-Immunoprecipitation Assay, Phospho-proteomics, Expressing, Activation Assay

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 silencing enhances cisplatin-induced apoptosis in bladder cancer cells. ( A ) FN1 knockdown efficiency in T24-R and UC3-R cells was confirmed by Western blot analysis. ( B ) FN1 knockdown efficiency in T24-R and UC3-R cells was confirmed by RT-qPCR analysis. ( C ) Silencing FN1 significantly increased apoptosis in the resistant cell lines by TUNEL staining. ( D ) Silencing FN1 reduced the IC50 of cisplatin in the resistant cell lines by CCK-8 assay. ( E ) In resistant cells, FN1 knockdown induced the expression of pro-apoptotic mediators, including BAX and cleaved-caspase-3, but suppressed levels of the anti-apoptotic protein Bcl-2. ( F ) Cell apoptosis rates were quantified by flow cytometry. For all panels, a t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, ** p < 0.01

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, TUNEL Assay, Staining, CCK-8 Assay, Expressing, Flow Cytometry

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 silencing inhibits tumor growth in vivo , enhancing cisplatin sensitivity. ( A ) Representative images of tumors from each treatment group. ( B ) Tumor volume growth curves over time measured in the T24-R xenograft model. ( C ) Apoptosis in tumor tissues was detected by TUNEL staining. ( D ) Immunohistochemical analysis showed decreased FN1 expression in tumor sections from the FN1 knockdown group. For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. * p < 0.05, *** p < 0.001

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: In Vivo, TUNEL Assay, Staining, Immunohistochemical staining, Expressing, Knockdown

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 regulates FAK (Y397) phosphorylation and mediates cisplatin resistance in bladder cancer cells. ( A ) Silencing FN1 in resistant cell lines reduced the phosphorylation of FAK (Y397) as detected by Western blot. ( B ) T24 and UC3 parental and resistant cells were treated with a fixed dose of cisplatin along with a gradient of rFN1 for 48 h. Phosphorylation of FAK (Y397) was assessed by Western blot. Resistant cells (T24-R, UC3-R) showed sensitivity to rFN1 at lower concentrations under cisplatin stress. ( C ) Silencing FN1 in resistant cell lines reduced the phosphorylation of FAK (Y397) as detected by immunofluorescence. ( D ) rFN1 addition increased resistance index in resistant strains. ( E ) The addition of rFN1 reduced apoptosis in resistant strains. For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, *** p < 0.001

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Inhibition of calpain9 attenuates peritoneal dialysis-related peritoneal fibrosis

doi: 10.3389/fphar.2022.962770

Figure Lengend Snippet: Calpain activity and calpain nine expression is increased during peritoneal fibrosis. (A) . Western blotting (WB) showing the expression of Calpain9 (CAPN9), SBDP cleavage products (120 and 150 kD), Collagen I (Col I) and Fibronectin (FN) in visceral peritoneal lysates of untreated (Ctrl), 6-week PDF (F–I) or 21-day CG treated mice. (B–E) SBDP (120 and 150kD), FN, Col I, CAPN9 in the peritoneal were examined. GAPDH was detected as loading control. Values are the mean ± s. e.m. n = 6, * p < 0.05, ** p < 0.01, vs. control (Ctrl). (J) Isolation and identification of RPMC. Immunofluorescence staining was used to identify the isolated RPMC. Vimentin was labeled with red, cytokeratin 18 was stained with green, and the nucleus was stained with blue. Magnification objective, ×20. Scale bar: 50 μm. (K–O) Western blot was used to detect the expression of SBDP (120 and 150kD), FN, Col I, CAPN9 in total lysates of RPMC untreated (Ctrl) or treated with 10 ng/ml TGF-β1 for 48 h β-actin was detected as a control. Values are the mean ± s. e.m. n = 3, * p < 0.05,** p < 0.01, vs. control (Ctrl). (P) Increased expression of CAPN9 induced by TGF-β in RPMC(top) and HmrSV5 (bottom). The cells were fixed, permeable and closed and stained with monoclonal antibodies against α-SMA and CAPN9. Immunofluorescence inverted microscope showed that green represented α-SMA, and red represented CAPN9. Magnification objective, × 20. Scale bar: 50 μm.

Article Snippet: The same amount of total protein (20 μg) was separated by SDS/PAGE electrophoresis, 5% skimmed milk powder was blocked for 1 h and then transferred to nitrocellulose membrane, and anti-rat β-actin (Proteintech, United States), GAPDH(Proteintech, United States), Fibronectin (FN) (Proteintech, United States), Collagen-I (Col-I) (Boster, China), α-Spectrin (SBDP) (Millipore,United States), calpain9 (Proteintech, United States), active β-catenin (Cell Signaling Technology,United States), E-Cadherin (Cell Signaling Technology,United States), p Smad2/3(Cell Signaling Technology,United States), Smad2/3 (Affinity, China), Snail (Abcam, United Kingdom) primary antibodies, incubated overnight at 4°C, the next day after washing the membrane with TBST, incubate with the corresponding fluorescent secondary antibody (LICOR, United States) for 1 h at room temperature.

Techniques: Activity Assay, Expressing, Western Blot, Control, Isolation, Immunofluorescence, Staining, Labeling, Bioprocessing, Inverted Microscopy

Journal: Frontiers in Pharmacology

Article Title: Inhibition of calpain9 attenuates peritoneal dialysis-related peritoneal fibrosis

doi: 10.3389/fphar.2022.962770

Figure Lengend Snippet: Calpain9 deficiency attenuated TGF-β-induced ECM protein expression and EMT. (A–F) Western blot showed Fibronectin (FN), Collagen I (Col I), Calpain9 (CAPN9), E-Cadherin, p Smad2/3 and Smad2/3 expression in whole cell lysates with or without TGF-β, HmrSV5, NC control or CAPN9-targeted siRNA transfection. * p < 0.05, ** p < 0.01, vs. TGF-β group.# p > 0.05. vs negative control (NC).

Article Snippet: The same amount of total protein (20 μg) was separated by SDS/PAGE electrophoresis, 5% skimmed milk powder was blocked for 1 h and then transferred to nitrocellulose membrane, and anti-rat β-actin (Proteintech, United States), GAPDH(Proteintech, United States), Fibronectin (FN) (Proteintech, United States), Collagen-I (Col-I) (Boster, China), α-Spectrin (SBDP) (Millipore,United States), calpain9 (Proteintech, United States), active β-catenin (Cell Signaling Technology,United States), E-Cadherin (Cell Signaling Technology,United States), p Smad2/3(Cell Signaling Technology,United States), Smad2/3 (Affinity, China), Snail (Abcam, United Kingdom) primary antibodies, incubated overnight at 4°C, the next day after washing the membrane with TBST, incubate with the corresponding fluorescent secondary antibody (LICOR, United States) for 1 h at room temperature.

Techniques: Expressing, Western Blot, Control, Transfection, Negative Control

Journal: Molecules (Basel, Switzerland)

Article Title: Effects of the Calix[4]arene Derivative Compound OTX008 on High Glucose-Stimulated ARPE-19 Cells: Focus on Galectin-1/TGF-β/EMT Pathway.

doi: 10.3390/molecules27154785

Figure Lengend Snippet: Figure 7. EMT markers in ARPE-19 cells. (A) ACTA2 and (B) Fn1 mRNA and protein levels. ACTA2 and Fn1 relative mRNA levels are reported as 2ˆ−∆∆Ct ± SD and quantized by using GAPDH as control. ACTA2 and Fn1 protein levels are reported as ng/mL ± SD of 9 observations per group (three independent experiments, each done in triplicate). ** p < 0.01 vs. NG; ◦p < 0.05 and ◦◦p < 0.01 vs. HG; NG, normal glucose (5 mM) + vehicle; M, mannitol (30 mM); HG, high glucose (35 mM) + vehicle; OTX 2.5, OTX 2.5 µM; OTX 5, OTX 5 µM; OTX 10, OTX 10 µM.

Article Snippet: Competitive ELISA tests were used to quantify the cellular levels of human AP4S1 (My Biosource, MBS7206462), ACTA2 (or α-SMA) (antibodies-online, ABIN6953406), and Fn1 (Cusabio, CSB-E11850h).

Techniques: Control