Journal: JCI Insight

Article Title: Cytoskeleton-associated protein 4 affects podocyte cytoskeleton dynamics in diabetic kidney disease

doi: 10.1172/jci.insight.181298

Figure Lengend Snippet: CKAP4 gene expression was quantified with qPCR ( A ) and protein expression by Western blot ( B ) in cultured HGECs, HMCs, and HPODs, normalized against the respective GAPDH gene/protein levels. Immunogold electron micrograph of podocyte feet and major processes; red arrows point at gold particles ( C ). Immunofluorescence of PDIA3 (ER marker, red), CKAP4 (green), and DAPI (blue) showing expression of CKAP4 in the ER of HPODs. Scale bar, 10 μm. ( D ). Immunofluorescence of phalloidin (actin cytoskeleton, red), CKAP4 (green), and tubulin (blue) showing the localization of CKAP4 in relation to cytoskeleton components ( E ). A : 1-way ANOVA with multiple comparisons, n = 3 per cell type. B : Kruskal-Wallis plus Dunn’s post hoc test, n = 5 per cell type. One representative blot is shown. Error bars in both panels represent average ± SEM. * P < 0.05, ** P < 0.01. AU, arbitrary units; CKAP4, cytoskeleton associated protein 4; HMCs, human mesangial cells; HPODs, human podocytes; HGECs, human glomerular endothelial cells; PDIA3, protein disulfide-isomerase A3.

Article Snippet: The antibodies used were: Alexa Fluor 594 anti-phalloidin (actin microfilaments/stress fibers, A12381, Invitrogen), Alexa Fluor 647 anti-tubulin (microtubules, Abcam, ab195884), AMAb90988 for PDIA3 (Atlas antibodies), and HPA000792 for CKAP4 (Atlas antibodies).

Techniques: Gene Expression, Expressing, Western Blot, Cell Culture, Immunofluorescence, Marker

Journal: JCI Insight

Article Title: Cytoskeleton-associated protein 4 affects podocyte cytoskeleton dynamics in diabetic kidney disease

doi: 10.1172/jci.insight.181298

Figure Lengend Snippet: Immunofluorescence staining of the ER marker PDIA3 in scr control and CKAP4 KD HPODs. Scale bars: 10 μm. ( A ). Protein expression of PDIA3 was quantified in untreated control, scr control, and CKAP4 KD HPODs using Western blot ( B ). Protein expression of ER stress marker intact and cleaved ATF6α in untreated control, scr control, and CKAP4 KD HPODs was quantified by Western blot ( C ). The total protein blots used for normalization are shown below each Western blot, and the quantification graphs are shown on the left side of the panel. B : n = 4 per group. Tukey’s post hoc after 1-way ANOVA. Error bars represent average ± SEM. AU, arbitrary units; CKAP4, cytoskeleton associated protein 4; PDIA3, protein disulfide - isomerase A3; ER, endoplasmic reticulum; KD, knockdown; HPODs, human podocytes; scr, scrambled; ATF6α, activating transcription factor 6α.

Article Snippet: The antibodies used were: Alexa Fluor 594 anti-phalloidin (actin microfilaments/stress fibers, A12381, Invitrogen), Alexa Fluor 647 anti-tubulin (microtubules, Abcam, ab195884), AMAb90988 for PDIA3 (Atlas antibodies), and HPA000792 for CKAP4 (Atlas antibodies).

Techniques: Immunofluorescence, Staining, Marker, Control, Expressing, Western Blot, Knockdown

Journal: Redox Biology

Article Title: Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes

doi: 10.1016/j.redox.2019.101142

Figure Lengend Snippet: Validation of ELISA assay for specific detection of PDI . A. PDI concentration curves (0.375–3 ng/mL) were generated using purified PDI quantified by ELISA. Measurements were analyzed on 4 independent assays. Data represent mean ± SEM. B-C . Human cell lysates (HUVEC, HCT116 and HKE3) were (B) quantified by ELISA ( n = 4; mean ± SEM) and (C) submitted to protein separation by reducing SDS-PAGE and immunoblotted with anti-PDI ELISA's capture antibody or anti-β actin (loading control) ( n = 3). Uncropped western blots are shown in Supplementary . D. Cross-reactivity with other PDI family members was analyzed using recombinant PDI, ERp57 and ERp5 (1.5–3 ng/mL) quantified by ELISA. Measurements were analyzed on 4 independent assays. Data represent mean ± SEM. E. Plasma PDI immunoprecipitation. Platelet-poor plasma was diluted in lysis buffer and immunoprecipitated using rabbit anti-PDI antibody. Immunoblotting was performed using mouse anti-PDI (RL90) ( n = 3 from independent experiments). F. Detection of plasma PDI reductase activity (di-eosin-GSSG assay) by formation of the reduced fluorogenic product eosin-5-isothiocyanate-coupled reduced glutathione (EGSH). It was measured in platelet-poor plasma exposed or not to 60 μM of rutin. Bar graphs represent the percentage cleavage of di-eosin-GSSG as compared to total reductase activity – Control (100%) in 10 min. The main result in this case is the rutin-inhibitable fraction of di-eosin-GSSG reductase activity. Data represent mean ± SEM from 5 independent experiments. *p < 0.05; ***p < 0.001 vs. control (One-way ANOVA followed by Tukey's post test).

Article Snippet: Platelet-poor plasma (no dilution), recombinant PDI solutions (reduced, oxidized, S-nitrosylated), human cell lysates, conditioned medium, and recombinant Erp57 (OriGene-TP305940) and ERp5 (OriGene-TP301710) were analyzed.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Concentration Assay, Generated, Purification, SDS Page, Control, Western Blot, Recombinant, Clinical Proteomics, Immunoprecipitation, Lysis, Activity Assay, GSSG Assay

Journal: Science immunology

Article Title: PDIA3 epitope-driven immune autoreactivity contributes to hepatic damage in type 2 diabetes.

doi: 10.1126/sciimmunol.abl3795

Figure Lengend Snippet: Fig. 4. High-fat and high-fructose diet induces liver inflammatory response. (A) Surface staining of splenocytes, nodal immune cells, and hepato- cytes for PDIA3 protein. (B) Normalized MFI of surface PDIA3 expressed on nodal immune cells, splenocytes, pancreatic cells, hepatocytes, liver im- mune infiltrates, cardiomyocytes, and adipocytes harvested from control and HFHF mice. A statisti- cally significant difference in the PDIA3 HFHF/control ratio was observed. Values, from n = 4 biologically independent replicates, are reported as mean rela- tive expression ± SD. Data were statistically analyzed using a two-tailed paired Student’s t test. Signifi- cance levels are reported as *P < 0.05. (C) Sequence of the linear autoantibody epitope (PDIA3 residues 104 to 128) shown in yellow mapped onto a sur- face representation of the PDIA3-tapasin complex crystal structure PDB.3F8U, with PDIA3 shown in magenta and tapasin shown in gray. (D to K) Livers were harvested from C57BL/6 mice on a regular diet or an HFHF diet for 3 months. Livers were fixed in formalin and stained to detect (D and E) steatosis (oil red staining), (F to I) hepatocyte injury and immune cell infiltration, and (J and K) proliferation. Steatosis, cellular infiltration and proliferation, lob- ular inflammation, and hepatocyte injury (ballooning) were all significantly up-regulated in HFHF mice as compared with controls. Values, from n = 6 biolog- ically independent replicates, are reported as mean relative expression ± SD and were statistically ana- lyzed using a two-tailed unpaired Student’s t test. (L and M) Increased ALT levels were seen in mice on a 3-month HFHF diet concomitantly with a de- creased AST/ALT ratio (<1), which is indicative of NASH. Values from n = 13 biologically independent replicates are shown here, and data were statistically analyzed using a two-tailed unpaired Student’s t test.

Article Snippet: Human His-tag PDIA3 protein (Creative BioMart, catalog no. PDIA3-20H) was loaded 2 g per well on 4 to 12% NuPAGE SDS-PAGE followed by transfer onto nitrocellulose membrane (0.45 m).

Techniques: Staining, Control, Expressing, Two Tailed Test, Sequencing

Journal: Science immunology

Article Title: PDIA3 epitope-driven immune autoreactivity contributes to hepatic damage in type 2 diabetes.

doi: 10.1126/sciimmunol.abl3795

Figure Lengend Snippet: Fig. 7. Elevated anti-PDIA3 autoantibodies in the sera of patients diagnosed with T2D, autoimmune hepatitis, and autoimmune cholangitis. Sera, collected from 25 healthy age- and sex-matched controls, 48 patients with a diagnosis of T2D, 28 subjects with a diagnosis of AIH, and 18 subjects with PBC, were tested in ELISA against the full-length human PDIA3 protein. Antibody titers were quantified in each subject, and each dot symbol represents the average of three technical replicates. Statistical analysis for each serum and the representative binding curves are presented in fig. S7 and table S7. The statistical significance was determined with one-way ANOVA including mixed-effects model restricted maximum likelihood (REML) followed by multiple comparisons performed with uncorrected Fisher’s LSD test (***P < 0.0001, **P < 0.001, and *P < 0.01). For specificity testing, commer- cially available polyclonal anti-PDIA3 antibody was used as positive control, where- as PBS buffer served as negative control.

Article Snippet: Human His-tag PDIA3 protein (Creative BioMart, catalog no. PDIA3-20H) was loaded 2 g per well on 4 to 12% NuPAGE SDS-PAGE followed by transfer onto nitrocellulose membrane (0.45 m).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Binding Assay, Positive Control, Negative Control

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: ERp57 KO mice display severe structural ECM defects in knee joint cartilage High-magnification transmission electron microscopic (TEM) analysis of articular cartilage isolated from 18-week-old WT and ERp57 KO mouse knees. KO samples exhibit a significantly lower ECM density around chondrocytes with holes in the territorial/interterritorial matrix (marked with arrows) (A). In microphotographs of WT samples, an average of 96% of the total area was covered with dense matrix, compared to 79% in the KO (B). Statistical evaluation was performed with Student’s t test. Data are mean ± SD. ∗∗ represents a p -value of <0.01. N (number of animals per genotype) ≥ 4; n (number of analyzed images per genotype) = 8; scale bars = 1 μm.

Article Snippet: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Transmission Assay, Isolation

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: Primary ERp57 KO chondrocytes produce less fibrillar matrix than WT cells Transmission electron microscopic (TEM) analysis of micromass cultures of primary WT and ERp57 KO chondrocytes isolated from knee joints of newborn mice revealed fewer and shorter cartilage fibrils in KO samples compared to WT controls (A), although the cell number is comparable (B). In microphotographs of WT samples, an average of 37% of the total area was covered with fibrils, compared to 25% in the KO (C). Statistical evaluation was performed with Student’s t test. Data are mean ± SD. ∗∗ represents a p -value of <0.01. ns indicates non-significant p -values. N (number of animals per genotype) = 4; n (number of micromasses per genotype) ≥ 10; scale bars = 500 nm.

Article Snippet: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Transmission Assay, Isolation

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: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

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: 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: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

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: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

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: All samples were then decellularized, fixed, blocked and incubated overnight with primary antibodies against fibronectin (sc-8422, monoclonal, from mouse, 1:50 Santa Cruz, Dallas, USA) or Col II (MAB8887, Merck Darmstadt, Germany, 1:200) and ERp57 (Novus, Centennial, USA, NBP1-84796, 1:200).

Techniques: Recombinant, In Vitro, Cell Culture, 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: ERp57 KO mice display severe structural ECM defects in knee joint cartilage High-magnification transmission electron microscopic (TEM) analysis of articular cartilage isolated from 18-week-old WT and ERp57 KO mouse knees. KO samples exhibit a significantly lower ECM density around chondrocytes with holes in the territorial/interterritorial matrix (marked with arrows) (A). In microphotographs of WT samples, an average of 96% of the total area was covered with dense matrix, compared to 79% in the KO (B). Statistical evaluation was performed with Student’s t test. Data are mean ± SD. ∗∗ represents a p -value of <0.01. N (number of animals per genotype) ≥ 4; n (number of analyzed images per genotype) = 8; scale bars = 1 μm.

Article Snippet: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Transmission Assay, Isolation

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: Primary ERp57 KO chondrocytes produce less fibrillar matrix than WT cells Transmission electron microscopic (TEM) analysis of micromass cultures of primary WT and ERp57 KO chondrocytes isolated from knee joints of newborn mice revealed fewer and shorter cartilage fibrils in KO samples compared to WT controls (A), although the cell number is comparable (B). In microphotographs of WT samples, an average of 37% of the total area was covered with fibrils, compared to 25% in the KO (C). Statistical evaluation was performed with Student’s t test. Data are mean ± SD. ∗∗ represents a p -value of <0.01. ns indicates non-significant p -values. N (number of animals per genotype) = 4; n (number of micromasses per genotype) ≥ 10; scale bars = 500 nm.

Article Snippet: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Transmission Assay, Isolation

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: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

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: 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: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

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: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

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: Recombinant human ERp57 protein , Novus , Cat#NBP2-52140.

Techniques: Recombinant, In Vitro, Cell Culture, Staining