Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 5. Expression changes of key GBM proteins and their response to FN1 Variants in TBMN. Immunofluorescence experiments assessed the expression of Laminin α5β2, COL4A3/4/5 and Integrin β1 in the GBM. The findings revealed non-homogeneous linear structure expression of these key GBM proteins in the presence of FN1 variants, indicating their potential involvement in the pathogenesis of TBMN. HC = healthy control.

Article Snippet: The antibodies used were list below: anti-FN1 antibody (1:50, ab2413, Abcam), anti-Fibronectin-IST9 antibody (1:50, sc-59,826, Santa Cruz), anti-Human CD31 antibody (1:50, AF806, R&D Systems), antiIntegrin β1 antibody (1:100, #34971S, CST), anti-COL4A3 antibody (1:50, #7076, Chondrex), anti-COL4A4 antibody (1:50, #7073, Chondrex), anti-COL4A5 antibody (1:50, #7077, Chondrex), anti-Laminin α5 antibody (1:200, ab210957, Abcam), anti-Laminin β2 antibody (1:200, Abcam), anti-HA-Tag antibody (1:200, T0008, Affinity), anti-Integrin β1 antibody (1:50, A21234, Abclonal), anti-Laminin α5 antibody (1:50, MAB-1924, Millipore), anti-Laminin α5 antibody (1:50, NBP1–18714, Novus Biologicals), anti-Laminin β2 antibody (1:50, sc-59,980, Santa Cruz), anti-Laminin γ1 antibody (1:50, sc-17,751, Santa Cruz), donkey polyclonal secondary antibody to rabbit Alexa FluorTM 555 (1:400, A31572, Thermo Scientific), donkey polyclonal secondary antibody to sheep Alexa Fluor® 647 (1:400, ab150179, Abcam), goat polyclonal secondary antibody to mouse Alexa FluorTM 488 (1:400, A-11001, Thermo Scientific), goat polyclonal secondary antibody to rabbit Alexa Fluor® 555 (1:400, ab150078, Abcam), goat polyclonal secondary antibody to rat Alexa Fluor® 647 (1:400, ab150159, Abcam), DAPI (1:1000, C1002, Beyotime Biotechnology).

Techniques: Expressing, Immunofluorescence, Control

Journal: Circulation Research

Article Title: Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture

doi: 10.1161/CIRCRESAHA.120.317295

Figure Lengend Snippet: Protein interaction network analysis reveals numerous interactions of proteins that are differentially abundant in aortas of SR-uPA +/0 mice. A protein-protein relational network was built based on experimentally validated direct interactions. The network is comprised of 87 proteins, each portrayed as a circular node (all nodes are identified in Data Set V in the Data Supplement ). Key highly connected nodes (hubs) are labeled together with 2 members of the matrix metalloproteinase family of extracellular proteases and several extracellular matrix components. ACTB indicates beta actin; AGRN, agrin; BCAM, basal cell adhesion molecule; ELN, elastin; FBLN5, fibulin 5; FN1, fibronectin 1; HSPG2, heparan sulfate proteoglycan 2; LAMA5, laminin subunit alpha 5; LAMB2, laminin subunit beta 2; LAMC1, laminin subunit gamma 1; LTBP4, latent transforming growth factor binding protein 4; MMP2, matrix metalloproteinase 2; MMP3, matrix metalloproteinase 3; MYH9, myosin heavy chain 9; NID1, nidogen1; NID2, nidogen 2; and PLAU, urokinase-type plasminogen activator.

Article Snippet: Primary antibodies and dilutions were rabbit anti-mouse laminin α5 serum (clone 504; a gift from Dr Lydia Sorokin, Munster University; 1:1000), mouse monoclonal anti-mouse endostatin (a fragment of COL18A1; Santa Cruz Biotechnology sc-32720; 1:100), and rat monoclonal anti-HSPG2 (Abcam ab2501; 1:100).

Techniques: Labeling, Binding Assay

Journal: Circulation Research

Article Title: Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture

doi: 10.1161/CIRCRESAHA.120.317295

Figure Lengend Snippet: Representative peptographs of extracts of ruptured vs stable human plaque segments. A and B , Extracts of ruptured (red) and adjacent stable (blue) segments of 5 human carotid plaques were analyzed using the PROTOMAP protocol. The extracts were subjected to SDS-PAGE, and the gels were cut into 22 slices, each corresponding to a molecular weight range. After in-gel trypsin digestion, peptides were extracted, identified by tandem mass spectrometry, and spectral counts were aggregated over all 22 slices. A , Proteins with differential abundance of lower-molecular weight peptides in extracts of ruptured vs. stable segments: ceruloplasmin (CP), angiotensinogen (AGT), ITIH4 (inter-alpha-trypsin inhibitor heavy chain), GPLD1 (phosphatidylinositol-glycan-specific phospholipase D), FA5 (coagulation factor V), SERPIND1 (serpin family D member 1), LAMB2 (laminin subunit beta-2), and SVIL (supervillin). B , ECM (Extracellular matrix) proteins that are significantly less abundant in extracts of ruptured vs stable human plaque segments: ACAN (aggrecan core protein), FBLN5 (fibulin 5), FMOD (fibromodulin), HAPLN (hyaluronan and proteoglycan link protein 1), LAMA5 (laminin subunit alpha 5), MFAP4 (microfibril-associated glycoprotein 4), LTBP1 (latent-transforming growth factor beta-binding protein 1), and VCAN (versican). A and B , Horizontal bars in each peptograph portray the total spectral counts for protein-specific peptides in each of the 22 gel slices (mean±SEM; n=5). Gel-slice number is on the leftward y -axis; molecular weight of the gel slices (in kilodaltons) is on the rightward y -axis.

Article Snippet: Primary antibodies and dilutions were rabbit anti-mouse laminin α5 serum (clone 504; a gift from Dr Lydia Sorokin, Munster University; 1:1000), mouse monoclonal anti-mouse endostatin (a fragment of COL18A1; Santa Cruz Biotechnology sc-32720; 1:100), and rat monoclonal anti-HSPG2 (Abcam ab2501; 1:100).

Techniques: SDS Page, Molecular Weight, Mass Spectrometry, Glycoproteomics, Coagulation, Binding Assay

Journal: Communications Biology

Article Title: Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves

doi: 10.1038/s42003-019-0638-5

Figure Lengend Snippet: Evaluation of cell surface proteins and DNA damage in CHO cells post-detachment. a – c Detached cells, with the proposed method, devoted by S p ; trypsinization, S t , and the scraper method, S s , were lysed in SDS-PAGE sample buffer, and protein samples were extracted and analyzed by western blot ( a ). Relative protein quantities of ( b ) anti-leukemia inhibitory factor receptor (LIFR) and ( c ) integrin α5 were measured and protein quantities were normalized to β-actin. d Number of AP sites was measured by AP site counting kit for evaluation of DNA damage. As a positive control, some of the cells was irradiated with UV for 30 min in the extracted DNA by using a DNA extraction kit. Data are expressed as mean ± SD ( n = 4, * p < 0.05, ** p < 0.01, all p SW > 0.05)

Article Snippet: The membranes were gently incubated in a blocking solution, Blocking One (Nacalai Tesque, Kyoto, Japan), for 1 h. The membranes were incubated with the following primary antibodies at 4 °C for 16 h: 0.4 μg/mL rabbit anti-human LIFR polyclonal antibody (22779-1-AP; Proteintech, Rosemont, IL, USA), a 1000-fold dilution of rabbit anti-human Integrin α5 monoclonal antibody (#98204; Cell Signaling Technology, Boston, MA, USA), and a 1000-fold dilution of rabbit anti-mouse β-actin polyclonal antibody (#4967; Cell Signaling Technology).

Techniques: SDS Page, Western Blot, Positive Control, Irradiation, DNA Extraction