Journal: Scientific Reports

Article Title: The von Willebrand factor stamps plasmatic extracellular vesicles from glioblastoma patients

doi: 10.1038/s41598-021-02254-7

Figure Lengend Snippet: VWF is transported in circulating EVs from GBM patients. ( A ) Equal volume of protein lysates from EV fractions, obtained with SEC followed by ultracentrifugation, and their corresponding plasma, from one GBM patient and one healthy donor, were separated by SDS-PAGE and analyzed by immunoblot for VWF, FCN3, and HSP70. Blots are representative of n = 3. ( B ) mRNA expression of von Willebrand factor (VWF) and ficolin-3 (FCN3) in healthy subjects and GBM patients estimated from The Cancer Genome Atlas (TCGA, n > 500 for GBM patients). ( C ) Anomalies currently reported in VWF and FCN3 genes. ( D ) Equal volume of protein lysates from EV fractions (3 GBM and 3 Healthy) were separated by SDS-PAGE and analyzed by immunoblot for VWF, GM130 (putative intracellular membrane protein contaminant), and Apolipoprotein A1 (APOA1, plasmatic protein). Healthy donor plasma and total cell lysate from GBM cells serve as controls. Blots are representative of n > 7 individual samples. ( E ) Densitometric analysis was performed on 7 and 8 independent EV preparations from Healthy and GBM liquid biopsies, respectively. ( F ) Concentration of VWF in plasmatic EVs from healthy donors and GBM patients (n = 20). Mann–Whitney test, * p < 0.05, ** p < 0.01, *** p < 0.001. S.D. are shown in panel E and S.E.M. in panel F.

Article Snippet: EVs were separated and concentrated from 500 μl of plasma as described above and the pellet was directly lysed in boiling Laemmli for 10 min. Proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes and blotted with the following antibodies, all diluted at 1/1000: CD9 (EXOAB-CD9A-1, SBI), CD63 (EXOAB-CD63A-1, SBI), VWF (sc-53466, Santa Cruz), FCN3 (CL7767AP, Cedarlane) , Apolipoprotein A1 (sc-376818, Santa Cruz), HSP70 (EXOAB-HSP70A, SBI), and GM130 (ab52649, Abcam) diluted at 1/1000.

Techniques: Clinical Proteomics, SDS Page, Western Blot, Expressing, Membrane, Concentration Assay, MANN-WHITNEY

Journal: Journal of Microbiology and Biotechnology

Article Title: Korean Red Pine ( Pinus densiflora ) Bark Extract Attenuates Aβ-Induced Cognitive Impairment by Regulating Cholinergic Dysfunction and Neuroinflammation

doi: 10.4014/jmb.2207.07015

Figure Lengend Snippet: ( A ) Western blot band image, Protein expression levels of ( B ) p-CREB, and ( C ) BDNF. The results shown are mean ± SD ( n = 3). Data were statistically considered at p < 0.05, and different small letters represent statistical difference.

Article Snippet: Brain-derived neurotrophic factor (BDNF) (CSB-PA05775A0Rb) was purchased from Cusabio Biotech (China), and anti-phospho-cAMP response element-binding protein (p-CREB) (9198) and anti-rabbit (7074S) were purchased from Cell Signaling Technology (USA).

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: Tissue Plasminogen Activator Neurotoxicity is Neutralized by Recombinant ADAMTS 13

doi: 10.1038/srep25971

Figure Lengend Snippet: ( A ) Representative immunoblots and quantitative analysis of the calpain- (145/150 kDa) and caspase-specific fodrin cleaved products (120 kDa) in sham-operated mice, and mice treated with vehicle, tPA, tPA in combination with rADAMTS 13, and rADAMTS 13 24 hours after MCA occlusion. The gels have been run under the same experimental conditions. ( B ) Six fields selected for immunohistochemical quantification of LC3, TUNEL, and FJB-labeled cells in the peri-infarct area in Nissl-stained coronal sections in all the groups. ( C ) Representative photomicrographs of TUNEL staining and quantification of TUNEL-positive cells in the ischemic brain of mice treated with vehicle, tPA, rADAMTS 13 in combination with tPA, and rADAMTS 13 24 hours after MCA occlusion. ( D ) Representative photomicrographs of Fluoro-Jade B (FJB) staining and quantification of FJB-positive cells in the ischemic brain of mice treated with vehicle, tPA, rADAMTS 13 in combination with tPA, and rADAMTS 13 24 hours after MCA occlusion. ( E ) Representative immunoblots of LC3 and quantification of the LC3-II/LC3-I ratio in sham-operated mice, and mice treated with vehicle, tPA, tPA in combination with rADAMTS 13, and rADAMTS 13 24 hours after MCA occlusion. The gels have been run under the same experimental conditions. ( F ) Representative photomicrographs of LC3 staining and quantification of LC3-positive cells in the ischemic brain of mice treated with vehicle, tPA, rADAMTS 13 in combination with tPA, and rADAMTS 13 24 hours after MCA occlusion. Bar = 30 μm. Values are mean ± SD (n = 5 per group). *P < 0.05.

Article Snippet: The primary antibodies were rabbit anti-LC3 (Light Chain 3, 4108), rabbit anti-pERK1/2 (phosphorylation of extracellular signal-regulated kinase1/2, 9101), rabbit anti-pJNK (c-Jun N-terminal kinases, 9251), rabbit anti-p-p38 (9211), rabbit anti-NR1 (N-methyl-D-aspartate receptor, 4204), rabbit anti-NR2A (4205), rabbit anti-NR2B (4207), rabbit anti-β-actin (4967) (all from Cell Signaling Technology, Beverly, MA, USA), mouse anti-α-fodrin (AA6, Enzo Life Sciences, Inc. Farmingdale, NY, USA), rabbit anti-tPA (ASHTPA-GF-HT, Molecular Innovations Inc., Novi, MI, USA).

Techniques: Western Blot, Immunohistochemical staining, TUNEL Assay, Labeling, Staining

Journal: Arthritis Research & Therapy

Article Title: Proteoglycan 4 downregulation in a sheep meniscectomy model of early osteoarthritis

doi: 10.1186/ar1898

Figure Lengend Snippet: Regional immunolocalisation of proteoglycan 4 (PRG4) with polyclonal antibody 06A10 in ovine tibial plateau cartilage. Lateral and medial tibial plateaux (LTP and MTP, respectively) from regions covered (COV) or uncovered (UNCOV) by the meniscus in sham-operated and meniscectomised (MEN) joints are shown, including enlarged views of respective surface zones as insets. Representative PRG4-positive (arrows) and PRG4-negative (asterisks) chondrocytes are indicated. Scale bar, 150 μm.

Article Snippet: Primary antibody incubations were performed overnight at 4°C with a 1:600 dilution of 06A10, a protein A purified rabbit polyclonal anti-PRG4 antibody generated by immunisation with a truncated form of recombinant human PRG4 (generously provided by Wyeth Pharmaceuticals, Boston, MA, USA).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Proteoglycan 4 downregulation in a sheep meniscectomy model of early osteoarthritis

doi: 10.1186/ar1898

Figure Lengend Snippet: Quantitative real-time PCR. (a) Topographical variation in proteoglycan 4 (PRG4) expression in sham-operated ('normal') joints as demonstrated by the ratio of mRNA copy numbers from cartilage normally covered (COV) versus cartilage normally uncovered (UNCOV) by menisci; in each case the average value is indicated by the line ( n = 6). The dotted horizontal line represents the expected ratio of 1 if there were no difference between regions. (b) PRG4 mRNA copy numbers (means ± SEM) from ovine articular cartilage after lateral meniscectomy (MEN). LTP, lateral tibial plateau; MTP, medial tibial plateau.

Article Snippet: Primary antibody incubations were performed overnight at 4°C with a 1:600 dilution of 06A10, a protein A purified rabbit polyclonal anti-PRG4 antibody generated by immunisation with a truncated form of recombinant human PRG4 (generously provided by Wyeth Pharmaceuticals, Boston, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing