Journal: International Journal of Molecular Sciences

Article Title: Insight into the Interactome of Intramitochondrial PKA Using Biotinylation-Proximity Labeling

doi: 10.3390/ijms21218283

Figure Lengend Snippet: Protein kinase A (PKA) catalytic subunit α is localized within mitochondria of HeLa cells. ( A ) Representative immunoblots ( n = 3) of PKA catalytic (cat) subunit, PKA regulatory (reg) subunit, the mitochondrial protein SDHa and of the cytosolic protein α-Tubulin with corresponding total protein load (TPL) in total cell lysate (TCL), cytosolic (Cyto) and mitochondrial (Mito) fractions isolated from HeLa cells. ( B ) Representative immunoblots ( n = 3) of PKA cat, PKA reg, SDHa and TOM20 with corresponding total protein load (TPL) in mitochondrial fractions isolated from HeLa cells and treated as indicated. ( C ) Representative micrographs ( n = 6) of HeLa cells expressing empty vector (pcDNA), PKA-Myc or mt-PKA-Myc and labelled with anti-Myc (green) and anti-TOM20 (red) as a mitochondrial marker. Scale bars = 5 μm. ( D ) Representative immunoblots ( n = 3) of Myc, SDHa and α-Tubulin with corresponding total protein load (TPL) in TCL, cyto and mito fractions isolated from HeLa cells expressing empty vector (pcDNA), PKA-Myc or mt-PKA-Myc. ( E ) Representative immunoblots ( n = 3) of Myc, TOM20, SDHa and SOD2 with corresponding total protein load (TPL) in mitochondrial enriched fractions isolated from HeLa cells expressing empty vector (pcDNA), PKA-Myc or mt-PKA-Myc and treated as indicated. ( F ) Representative immunoblots ( n = 3) of Myc, PKA Cat, ATP5a, MFN2 and ERp57 with corresponding TPL in pellet and supernatant (SN) obtained after treatment of mitochondria isolated from HeLa cells expressing PKA-Myc or mt-PKA-Myc and treated as indicated. ( G ) Representative immunoblots ( n = 5) of phospho-PKA substrates and Myc with corresponding total protein load (TPL) in mitochondrial enriched fractions isolated from HeLa cells expressing empty vector (pcDNA), PKA-Myc or mt-PKA-Myc.

Article Snippet: Protein immunodetection was performed using primary antibodies directed against PKA C-α (Cell Signaling; 4782S), PKA RI-α/β (Cell Signaling; 3927), Phospho-PKA Substrate (Cell Signaling; 9624S), α-Tubulin (Cell Signaling; 2144) SDHa (Abcam; ab14715), SOD2 (Santa Cruz; sc-30080), NDUF9a (Abcam; ab14713), Cytochrome c (Abcam; ab110325), TOM20 (Santa Cruz; (F-10) sc-17764), UQCRC2 (Abcam; ab14745), PAM16 (Abcam; Ab184157), Hsp60 (Santa Cruz; sc-13115), Hsp70 (Santa Cruz; sc-24), V5 (Cell Signaling; 13202S), Myc (Cell Signaling; 2276S), ATP5a (Abcam; ab14730), MFN2 (Cell signaling; 9482S), ERp57 (R&Dsystems; AF8219).

Techniques: Western Blot, Isolation, Expressing, Plasmid Preparation, Marker

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: eLife

Article Title: Co-movement of astral microtubules, organelles and F-actin by dynein and actomyosin forces in frog egg cytoplasm

doi: 10.7554/eLife.60047

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-PDIA3 (Rabbit polyclonal) , Boster Bio , Cat#: PB9772 , IHC (1:1000).

Techniques: Labeling, Recombinant, Expressing, Software