Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Effects of Insulin-Producing Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Type 1 Diabetes Mouse Model

doi: 10.3390/ijms23136877

Figure Lengend Snippet: Insulin secretion from pancreatic islets in hUC-IPCs and hAD-IPCs, as evaluated using the GSIS test. Low- (2.8 mM) and high-glucose (17.5 mM) conditions were used to stimulate hUC-IPCs and hAD-IPCs. Insulin secretion was quantitatively measured using insulin enzyme-linked immunosorbent assay (ELISA). Data are presented as the mean ± standard error of mean. ( n = 3 per IPC type; hUC-MSCs vs. hAD-MSCs, ** p < 0.01; *** p < 0.005). Abbreviations: LG, low glucose; HG, High glucose; GSIS, glucose-stimulated insulin secretion; hUC-IPCs, insulin-producing cells from human umbilical cord-derived mesenchymal stem cells; hAD-IPCs, insulin-producing cells from human adipose-derived mesenchymal stem cells.

Article Snippet: Insulin levels were measured in the supernatant after 1 h. Insulin secretion was determined using the human insulin ELISA Kit (Cat# ab278123; Abcam, Cambridge, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: medRxiv

Article Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

doi: 10.1101/2020.03.29.20041962

Figure Lengend Snippet: The N proteins of SARS-CoV, MERS-CoV and SARS-CoV-2 bind to MASP-2. (A) Lysates of 293T cells transfected with indicated plasmids were subjected to anti-Flag immunoprecipitation in the presence of 2 mM CaCl 2 or 1 mM EDTA, and analyzed by immunoblotting. IgG immnuoprecipitates were used as a negative control. (B) Lysates of 293T cells transfected with indicated plasmids were mixed with human serum (HS) and mouse serum (MS) and subjected to immunoprecipitation with anti-Flag agarose beads. The absorbates were probed with indicated antibodies. Purified recombinant MASP-2 was loaded as a marker. (C)-(E) Lysates of 293T cells transfected with indicated plasmids were subjected to anti-Flag immunoprecipitation in the presence of 2 mM CaCl 2 and analyzed by immunoblotting.

Article Snippet: Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.

Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, Purification, Recombinant, Marker

Journal: medRxiv

Article Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

doi: 10.1101/2020.03.29.20041962

Figure Lengend Snippet: The N proteins induce MASP-2 auto-activation and C4 cleavage. (A) Lysates from cells expressing indicated proteins were mixed with purified N or MBL and subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. (B) Purified MASP-2-Flag was incubated with/without N, MBL, and mannan at 37°C for 12 hr. Cleaved MASP-2 was probed with an anti-Flag antibody. (C) Purified MASP-2 and N proteins with/without anti-N monoclonal antibody were incubated with pre-conjugated MBL in mannan-coated plates at 4°C. Binding of MASP-2 was detected with an anti-MASP-2 antibody. (D) C4 was incubated with indicated components at 37°C for 0, 2, 6, 12 and 24 hr. C4 and a cleaved C4alpha’ fragment were detected with anti-C4alpha chain antibody. (E) C4 was incubated with indicated components at 37°C for 1 hr. C4 cleavage was detected as mentioned above. Ab, anti-MASP-2 antibody. (F)-(H) C4b deposition in relation to the concentration of N proteins of SARS-CoV, HCoV-229E, SARS-CoV-2, and MERS-CoV.

Article Snippet: Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.

Techniques: Activation Assay, Expressing, Purification, Immunoprecipitation, Western Blot, Incubation, Binding Assay, Concentration Assay

Journal: medRxiv

Article Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

doi: 10.1101/2020.03.29.20041962

Figure Lengend Snippet: N proteins potentiate LPS-induced pneumonia in mice. (A) BALB/c mice (10 mice/group) were infected with 1×10 9 PFU Ad-SARS N/Ad-null or a saline control, and LPS (5 mg/kg) was given on the 6th days post infection (d.p.i.). Antibodies (200 μg/kg) or C1INH (4 mg/kg) was injected 30 min before LPS injection. The fatality of mice was noted. (B) Lung paraffin sections were analyzed by HE staining. (C) Mice were infected with 1×10 8 PFU Ad-SARS N/Ad-null, and LPS (5 mg/kg) was given by on 6 d.p.i. Mice were sacrificed after LPS challenged for 6 hr. Frozen lung sections were stained with anti-C4b antibody. (D) SARS-CoV N and MASP-2 complex formation in frozen lung sections was measured by in situ PLA, as indicated by the red signals. Deposited C3 fragments were stained with FITC-labeled anti-C3c antibodies (green). Scale bar, 50 μm. (E) Mice were pre-infected with 1×10 9 PFU Ad-MERS N/Ad-null and treated with LPS, anti-MASP-2 antibody or C1INH as mentioned above. The mice survival curve was plotted. (F) Masp2 -/- and Masp2 +/+ C57BL/6N mice were infected N-expressing adenovirus and injected with LPS as mentioned above. The mice survival curve was plotted.

Article Snippet: Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.

Techniques: Infection, Injection, Staining, In Situ, Labeling, Expressing

Journal: medRxiv

Article Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

doi: 10.1101/2020.03.29.20041962

Figure Lengend Snippet: Schematic representation of MBL pathway over-activated by N protein of SARS/ MERS-CoV or SARS-CoV-2. (A) Virus binds to cell surface and S protein actives MBL. (B) Virus enters cells and expresses viral proteins including N protein. (C) N proteins release by secretion or after cells and virus lysis. (D) The extracellular soluble N protein dimmers interact with MASP-2, induce MASP-2 auto-activation and binding to MBL. (E) The accelerated activation of MASP-2 induces downstream complements cascades over-activation of the MBL pathway, and promotes cell lysis and N protein releases by secretion or complement mediated cytotoxicity, which may result in uncontrolled tissue damage and inflammation.

Article Snippet: Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.

Techniques: Lysis, Activation Assay, Binding Assay, Flow Cytometry Complement-Mediated Cytotoxicity Assay

Journal: Scientific reports

Article Title: Mannan-binding lectin serine protease-2 (MASP-2) in human kidney and its relevance for proteolytic activation of the epithelial sodium channel.

doi: 10.1038/s41598-022-20213-8

Figure Lengend Snippet: Figure 1. Detection of MASP-2 protein variants in liver, kidney and urine. (A) Three distinct murine monoclonal anti-human antibodies targeting MASP-2 were used, two reacting with epitopes within the SP-domain of MASP-2; an in-house (15-7-3) and a commercial (Origene), the third (13B7) reacting with an epitope in the N-terminal domain. The six domains of intact MASP-2 are illustrated, including the two CUB-domains, the epidermal growth factor (EGF)-like domain, the two complement control repeats (CCPs) and the serine protease (SP) domain. The alternatively spliced short variant MAp19 also shown. N- and C-terminals are outlined. (B) Predicted cleavage products detected by the two antibodies. Under non-reduced conditions, the antibody targeting the A-chain (13B7) would detect bands at ≈ 75 kDa (MASP-2) and ≈ 20 kDa (MAp19), and the antibody targeting the B-chain (15-7-3) would detect at ≈ 75 kDa. Under reduced conditions, 13B7 is expected to detect the 47 kDa A-chain if MASP-2 is activated, whereas 15-7-3 antibody is expected to detect the 27 kDa variant (the SP-domain) (Fig. 2B, lane 3 + 4). (C) When using the anti-SP-antibody (15-17-3), a band ≈ 70 kDa was detected in human kidney homogenate in both human pools of kidney cortex (HCP, n = 4), outer (HOM, n = 4) and inner medulla (HIM, n = 4) in reduced samples. (D). A similar band was detected in human liver homogenate, under reduced conditions, when using the anti-SP-antibody (15-17-3). HCP = human cortex pool (n = 4). (E) In human plasma, the anti-SP-antibody (15-17-3) detected a band migrating at ≈ 75 kDa. (F) Full length MASP-2 was not detected with protein from urinary extracellular vesicles (uEV) or in experimentally concentrated urine when using the 15-17-3 antibody. When testing the antibody targeting the A-chain (13B.7), no signal was detected in a kidney cortex pool (HCP, n = 4). In contrast, the alternative splice variant MAp19 was detected in uEVs and concentrated urine. (G) Under non-reduced condition, a band likely representing intact MASP-2 at 75 kDa was apparent in kidney cortex (HCP) and plasma with the in house 15-17-3 antibody whereas the commercial monoclonal antibody (OriGene #TA812533S) directed against the SP domain confirmed the presence of a 75 kDa band in non-reducing lanes. Both antibodies detected a band migrating slightly faster, at ≈ 70 kDa, in a human kidney cortex pool (HCP) and plasma. (H) Both the commercial antibody (OriGene #TA812533S) and the in-house monoclonal antibody (15-17-3) detected a band ≈ 75 kDa, when recombinant MASP-2 was applied to the membrane under non-reduced conditions. Original uncropped blots are presented in Supplementary Fig. S2A–F.

Article Snippet: : - + - + - + - + 100 - 75 - 50 - 37 - 25 - 150 - 100 - 75 - 50 - 37 - 25 - 150 - kDa:kDa: 15-17-3OriGene TA812533S HCP 20 μg Plasma Plasma 75 - 37 - 50 - 100 - OriGene TA812533S 15-17-3 Recombinant MASP-2 μL 5 2.5 1.25 0.63 5 2.5 1.25 0.63 H 15-17-3 25 - 20 - 13B7 HC P uE V Ur in e OriGene TA812533S μg: kDa: M A SP -2 nitca-β Theoretical migratory pattern Figure 1.

Techniques: Control, Variant Assay, Clinical Proteomics, Recombinant, Membrane

Journal: Molecular Biology Reports

Article Title: Association between mannose binding lectin gene polymorphisms and clinical severity of COVID-19 in children

doi: 10.1007/s11033-023-08524-z

Figure Lengend Snippet: Serum MBL and MASP-2 levels according to the clinical features of the patients

Article Snippet: The human MBL and MASP-2 ELISA kits measured serum MBL and MASP-2 levels in samples from patients (BT LAB, China).

Techniques:

Journal: Molecular Biology Reports

Article Title: Association between mannose binding lectin gene polymorphisms and clinical severity of COVID-19 in children

doi: 10.1007/s11033-023-08524-z

Figure Lengend Snippet: Serum MBL and MASP-2 levels, according to the Codon 54 polymorphisms

Article Snippet: The human MBL and MASP-2 ELISA kits measured serum MBL and MASP-2 levels in samples from patients (BT LAB, China).

Techniques: