antibodies against fibronectin  (Proteintech)

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Cultured C28/I2 ERp57 KO cells exhibit a reduced extracellular network of fibronectin 1 but unchanged collagen II fibrils Immunofluorescence analyses of the extracellular matrix (ECM) produced by C28/I2 WT and C28/I2 ERp57 KO chondrocytes, examined after fixation (Cells + Matrix) or after decellularization and fixation (Matrix) to visualize the ECM fibrils without cell-derived signals. The figure shows the projections of z-stacks. Punctate Col II signals in non-decellularized samples (Cells + Matrix) reveal Col II-containing vesicles near/above the nuclei of chondrocytes. Fibronectin (FN1) and collagen II (Col II) fibrils were detected in WT samples, including cells and matrix, and also in decellularized samples containing only matrix. The FN1 network was significantly reduced in KO samples (A). In contrast, the Col II network was comparably well developed in ERp57 KO and WT cells (B). Quantitative analysis of the decellularized samples revealed a reduction in the mean staining intensity of the FN1 matrix by more than 60% in the KO samples compared to WT controls and no statistically significant difference in Col II staining in samples of both genotypes. (C) Statistical evaluation was performed with the Student’s t test. Data are mean ± SD. ∗∗∗∗ represents a p -value of <0.0001, ns indicates non-significant p -values. N ≥ 8 (number of experiments), n ≥ 30 (technical replicates). Scale bars = 20 μm.

Article Snippet: To analyze FN1 in the newly generated ECM, antibodies against fibronectin (sc-8422, monoclonal, from mouse, Santa Cruz, Dallas, USA, 1:50 or F3648, polyclonal, from rabbit, Merck Darmstadt, Germany, 1:200) were used.

Techniques: Cell Culture, Immunofluorescence, Produced, Derivative Assay, Staining

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: The formation of disulfide bridges is essential for fibronectin 1 but not for collagen II fibrillogenesis Immunofluorescence analyses of FN1 and Col II fibrils produced by C28/I2 WT chondrocytes cultured for 72 h in the presence of non-cell-permeable thiol-blocking agents p -Chloromercuriphenylsulfonate (pCMPS) (1.5, 5, and 15 μM) (A) and Monobromo(trimethylammonio)bimanbromide (QBBR) (60, 300, and 600 μM) (B) to inhibit extracellular protein disulfide isomerase activity. The samples were examined after decellularization and fixation. The quantitative analysis of the mean staining intensities of the fluorescence signals revealed a reduction of (long) FN1 fibrils with increasing concentrations of both thiol-blocking agents, while the Col II network was not impaired (C). Statistical evaluation was performed with ordinary one-way ANOVA with Tukey’s post-hoc-test. Data are mean ± SD. ∗∗∗∗ represents a p -value of p < 0.0001 ∗∗∗ represents a p -value of p < 0.001, ∗∗ represents a p -value of p < 0.01, ns indicates non-significant p -values. N = 3 (number of experiments), n ≥ 8 (technical replicates). Scale bars = 20 μm.

Article Snippet: To analyze FN1 in the newly generated ECM, antibodies against fibronectin (sc-8422, monoclonal, from mouse, Santa Cruz, Dallas, USA, 1:50 or F3648, polyclonal, from rabbit, Merck Darmstadt, Germany, 1:200) were used.

Techniques: Immunofluorescence, Produced, Cell Culture, Blocking Assay, Activity Assay, Staining, Fluorescence

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Extracellular ERp57 colocalizes with fibronectin 1 fibrils Co-Immunofluorescence analysis of FN1/ERp57 (A, top panel) and Col II/ERp57 (B, bottom panel) on decellularized matrices. In C28/I2 WT samples, ERp57 was detected on FN1 fibrils in different quantities (← ERp57 high, FN1 high, < ERp57 high, FN1 low, ∗ ERp57 low, FN1 high). The Col II network showed no direct colocalization with ERp57, however ERp57 was detectable in close vicinity to Col II structures (◄). N = 3. Scale bars = 20 μm.

Article Snippet: To analyze FN1 in the newly generated ECM, antibodies against fibronectin (sc-8422, monoclonal, from mouse, Santa Cruz, Dallas, USA, 1:50 or F3648, polyclonal, from rabbit, Merck Darmstadt, Germany, 1:200) were used.

Techniques: Immunofluorescence

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Extracellular ERp57 interacts directly with fibronectin 1 fibrils Proximity ligation assays (PLA) showed FN1/ERp57 interactions, visible as red dots on fibrillar structures of the extracellular matrix (ECM) (A). The corresponding statistical analysis (B) revealed a mean staining intensity of 0.284 ± 0.1065, which differed significantly from the mean staining intensities in the matrix of ERp57 KO cells and in the negative control (WT matrix without primary antibodies). In contrast, no interactions between Col II and ERp57 were detectable using PLA. The mean staining intensity in the WT-produced ECM did not exceed the background staining of the fibrils produced by ERp57 KO cells or the negative control (WT matrix without both primary antibodies). Short-term incubation with the reducing agent dithiothreitol (DTT) reduced PLA signals (C and D) significantly. Omission of ERp57 or FN1 antibodies reduced PLA signals to background levels (D). Statistical evaluation was performed with one-way ANOVA with Tukey’s post-hoc-test. Data are mean ± SD. ∗ represents a p -value of <0.05. N = 3 (number of experiments), n = 12 (technical replicates). Scale bars = 20 μm.

Article Snippet: To analyze FN1 in the newly generated ECM, antibodies against fibronectin (sc-8422, monoclonal, from mouse, Santa Cruz, Dallas, USA, 1:50 or F3648, polyclonal, from rabbit, Merck Darmstadt, Germany, 1:200) were used.

Techniques: Ligation, Staining, Negative Control, Produced, Incubation

Journal: iScience

Article Title: Extracellular ERp57 promotes fibronectin fibril formation during matrix assembly of articular cartilage

doi: 10.1016/j.isci.2025.114046

Figure Lengend Snippet: Active recombinant ERp57 protein added to the culture medium increases the fibronectin 1 fibrillogenesis around ERp57 KO cells in vitro Immunofluorecence analysis of FN1 and Col II on decellularized matrices of C28/I2 WT and C28/I2 ERp57 KO cells. Some of the KO cells were cultured for the entire culture period of 72 h in the presence of 0.1 μM active recombinant ERp57 protein or in the presence of 0.1 μM active recombinant ERp57 protein with the addition of 5 μM p -Chloromercuriphenylsulfonate (pCMPS) or 300 μM Monobromo (trimethylammonio) bimanbromide (QBBR) (A). KO cells showed in the quantitative analysis a strongly reduced staining intensity of FN1 and an unchanged staining intensity of Col II (B). The addition of active recombinant ERp57 protein to the cell culture medium of KO cells led to an increase in the mean staining intensity of FN1 (partial rescue), which was reduced again by the simultaneous addition of pCMPS and QBBR. The staining intensity of Col II was not significantly affected by the addition of active recombinant ERp57 protein in the presence or absence of pCMPS or QBBR. Statistical evaluation was performed with two-way ANOVA with Tukey’s post-hoc-test. Data are mean ± SD. ∗∗∗∗ represents a p -value of p < 0.0001, ∗∗ represents a p -value of p < 0.01, ns indicates non-significant p -values. N ≥ 5 (number of experiments), n ≥ 16 (technical replicates). Scale bars = 20 μm.

Article Snippet: To analyze FN1 in the newly generated ECM, antibodies against fibronectin (sc-8422, monoclonal, from mouse, Santa Cruz, Dallas, USA, 1:50 or F3648, polyclonal, from rabbit, Merck Darmstadt, Germany, 1:200) were used.

Techniques: Recombinant, In Vitro, Cell Culture, Staining