α 2 macroglobulin antibodies  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

doi: 10.3389/fimmu.2020.575607

Figure Lengend Snippet: Human platelets contain proBDNF. (A) ProBDNF immunoblotting of human washed platelet lysates (15 µg) from six different healthy volunteers. Recombinant proBDNF (3 ng) and human cortex lysate (3 µg) were used as positive controls. Molecular weight is indicated on the left (kDa) and primary antibody on the right. Experiments representative of n=9 for R-176 and n=10 for mab31751 antibody. IB, immunoblotting. (B) Immunoblotting of proBDNF and BDNF in different fractions of washed human platelets. P-Selectin was used as control protein in the membrane fraction, p65 NF-ĸB was used as control protein in the cytosol, and α-tubulin was used as control protein in the cytoskeleton. The equivalent of the protein content of 3 x 10 7 platelets was loaded for each fraction on the gel. Representative experiment of n=4 different volunteers. (C) ProBDNF treatment with PNGase F in washed human platelet lysates. U87-MG glioblastoma cells were used as a control. rProBDNF, recombinant proBDNF (3 ng); cortex, human cortex lysate (3 µg); platelets, whole human platelet lysate (representative experiment of n=4 different volunteers, 7.5 x 10 8 platelets per well); −PNGF, platelets treated with GlycoBuffer, and incubated at 37°C for 60 min without PNGase F; +PNGF, platelets treated with GlycoBuffer and incubated at 37°C for 60 min with PNGase F; PNGF alone, PNGase F incubated at 37°C for 60 min without platelet lysate. CD42b and sortilin were used as controls of protein deglycosylation in platelets and in U87-MG cells, respectively. n=3 different volunteers for PNGase treatments in human platelets and n=4 independent experiments for U87-MG cells. (D) Representative flow cytometry experiment showing surface and intracellular proBDNF in human washed platelets and in U87-MG and U251-MG glioblastoma cell lines. Mouse IgG 2b was used as isotype control. Percentage of expression are indicated on the figure. n=10 different healthy volunteers for human platelets; n=3 independent experiments for each glioblastoma cell line. (E) Confocal microscopy imaging of proBDNF in human permeabilized washed platelets (top) and in permeabilized U-251 MG cells (bottom). Mouse IgG 2b was used as isotype control. ProBDNF was labelled using Alexa488 fluorochrome (in green). Nuclei were stained with DAPI (in blue). Scale bar = 2 µm and 200 µm for washed platelets and U-251 MG cells images, respectively. (F) Immunoblotting of α 2 -macroglobulin at increasing quantities of loaded proteins (1–20 µg) obtained from a washed platelet lysate or platelet-poor plasma (PPP) from the same individual (#23). α-tubulin was used as loading control. Molecular weight is indicated on the left (kDa) and primary antibody on the right. PPP, platelet poor plasma; PLTs, platelets; IB, immunoblotting.

Article Snippet: Protein samples were separated on a 12% SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto a PVDF 0.2 μm membrane and fixed with glutaraldehyde 0.5% for 30 min. Membranes were washed three times with TBS-T (Tris-buffered saline, 0.1% Tween 20) for 10 min and blocked for 1 h at RT in blocking solution [3% bovine serum albumin (BSA) in TBS-T or 5% milk in TBS-T] before incubation with anti proBDNF or anti α 2 -macroglobulin antibodies (Biosensis, anti-proBDNF R-176 polyclonal rabbit antibody, 0.25 μg/ml; R&D systems, anti-proBDNF mab31751 monoclonal mouse antibody, clone 584412, 0.5 μg/ml; Abbexa, anti-α 2 -macroglobulin abx132389, monoclonal mouse antibody, 1 μg/ml) overnight at 4°C.

Techniques: Western Blot, Recombinant, Molecular Weight, Control, Membrane, Incubation, Flow Cytometry, Expressing, Confocal Microscopy, Imaging, Staining, Clinical Proteomics

Journal: Proteome Science

Article Title: Proteomic profiling for the identification of serum diagnostic biomarkers for abdominal and thoracic aortic aneurysms

doi: 10.1186/1477-5956-11-27

Figure Lengend Snippet: Examination of the decreased level of α-2-macroglobulin in postsurgical sera compared with that in presurgical sera of both AAA and TAA patients, and restoration of its level to that in normal control sera. Western blsot analyses with anti-α-2-macroglobulin antibody in presurgical (pre) and postsurgical (post) sera of 12 patients with AAA ( A ) and 10 patients with TAA ( B ) were performed as indicated by arrows. Representative photos of two each of patients (patient no. #AAA11, #AAA13, #TAA10 and #TAA11) as well as a mixture of 10 normal control sera (N) are shown. To confirm equal levels of proteins per lane, non-specific proteins stained with Coomassie Brilliant Blue are shown (CBB). The intensity of each band in presurgical and postsurgical sera of 12 AAA and 10 TAA patients and 10 normal control sera that reacted with anti-α-2-macroglobulin antibody was measured. Ratios of levels of α-2-macroglobulin in presurgical and postsurgical sera and normal control sera compared with that in the mixture of 10 normal control sera (1.0) were determined by band intensity. Means ± SE of triplicate experiments were calculated, and statistical analysis was performed using the paired t -test between presurgical and postsurgical data, whereas it was done using the unpaired t -test between postsurgical and normal control data. ** p < 0.01.

Article Snippet: The membranes were reacted with rabbit polyclonal anti-fibronectin antibody (Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-LRG1 antibody (Abnova, Taipei, Taiwan), rabbit polyclonal anti-α-2-macroglobulin antibody (Abcam, Tokyo, Japan), goat polyclonal anti-kallistatin antibody (R & D Systems, Minneapolis, MN, USA), mouse monoclonal anti-gelsolin antibody (Sigma-Aldrich, St. Louis, MO, USA) or goat polyclonal anti-afamin antibody (GeneTex, Irvine, CA, USA).

Techniques: Western Blot, Staining

Journal: Physiological Genomics

Article Title: Comprehensive genomic profiling in diabetic nephropathy reveals the predominance of proinflammatory pathways

doi: 10.1152/physiolgenomics.00028.2013

Figure Lengend Snippet: Verification of RNA-seq data. Upregulated genes, downregulated genes, and genes with unchanged expression in diabetic kidneys were randomly selected, and alterations in expression were evaluated by RT-PCR. The changes observed by RT-PCR were very tightly correlated (log-log transformation) with the results found by next-generation sequencing. Representative immunoblots also show that protein levels for selected transcripts were consistent with RNA-seq and RT-PCR data. CYP8B1: cytochrome p450, family 8, polypeptide 1. ACOX2: acyl-CoA oxidase 2. EGF: epidermal growth factor. CDCa7: cell division cycle associated 7. COL1a1: collagen type 1 alpha 1. KNG1: kininogen 1. CDKN1: cyclin-dependent kinase inhibitor 1. A2M: α-2-macroglobulin.

Article Snippet: Western Blot Proteins were separated from renal homogenates on a 12% acrylamide SDS-PAGE gel, electrophoretically transferred to a BIO-RAD Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) at 15 mA, and labeled with anti-α-2-macroglobulin (A2M) goat antibody (Cat. #M-0140; Sigma, St. Louis, MO), anti-collagen 1 rabbit antibody (Cat. #600-401-103s; Rockland, Gilbertsville, PA), anti-acyl-CoA oxidase 2, branched chain (ACOX) goat antibody (Cat. #PA5-18671; Thermo Scientific, Waltham, MA) and anti-actin mouse antibody (Cat. #Ab6276; Abcam, Cambridge, MA).

Techniques: RNA Sequencing Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Next-Generation Sequencing, Western Blot