Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: Specificity and potency of anti-PAR2 monoclonal antibody MEDI0618 . ( A ) Live staining of PAR2-expressing (1321N1-hPAR2.cl8) or non-expressing (1321N1 parental) cell lines with MEDI0618 directly conjugated to Alexa Fluor 647. Scale bar = 20 µm. ( B ) Flow cytometry of hPAR2 overexpressing cells (1321N1-hPAR2.cl8) or non-expressing cells (1321N1 parental cell line) or A549 cells endogenously expressing hPAR2 live labelled with MEDI0618. ( C and D ) Calcium imaging from 1321N1-hPAR2 cells pretreated with MEDI0618 hIgG or an isotype control protein. ( C ) Exemplar raw calcium trace from a single well pretreated with MEDI0618 or isotype control antibody both at 1 nM, followed by PAR2 agonist stimulation with matriptase (10 nM). ( D ) Antibody titration of the anti-PAR2 antibodies MEDI0618 or PAR650097 or an isotype control antibody. Data show the matriptase (10 nM)-mediated calcium signal after preincubation with antibody and normalized to the matriptase response in the absence of antibody treatment. ( E ) Calcium imaging from A549 cells pretreated with MEDI0618, isotype control protein or PAR1 inhibitors (ATAP2 + WEDE15 mAbs) followed by 10 nM thrombin (PAR1 agonist) stimulation. Data are presented as mean ± standard error of the mean, n = 4. The concentration of the inhibitor is shown on the x -axis. Data are normalized to the peak thrombin calcium response in the absence of inhibitor pretreatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Staining, Expressing, Flow Cytometry, Imaging, Control, Titration, Concentration Assay

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: PAR2 functional expression in human and mouse cells relevant to migraine . Whole well calcium imaging from primary human dural fibroblasts (HDuF), human dural microvascular endothelial (HDuMEC) and mouse brain endothelial (bEnd.3) cells. ( A , D and G ) PAR2 agonists concentration response curve in ( A ) HDuF, ( D ) HDuMEC and ( G ) bEnd.3 cells. ( B , E and F ) Effect of MEDI0618 and isotype control protein (IgG) on inhibition of matriptase-induced calcium signalling at 30 nM in ( B ) HDuF, ( E ) HDuMEC and ( H ) bEnd.3 cells. ( C , F and I ) Representative calcium imaging traces of 30 nM matriptase-evoked activity following MEDI0618 or isotype control protein preincubation in ( C ) HDuF, ( F ) HDuMEC and ( I ) bEnd.3 cells. ( J and K ) Single-cell calcium imaging from mouse trigeminal neuron cultures. ( J ) Pseudocolour images of fura-2 ratio intensity show a subset of trigeminal neurons activated by treatment with 10 µM LIGRLO in comparison to 20 mM KCl treatment. Scale bar = 20 µm. ( K ) Representation of fura-2 traces recorded from two individual neurons during acute LIGRLO (10 µM) or KCl (20 mM) treatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Functional Assay, Expressing, Imaging, Concentration Assay, Control, Inhibition, Activity Assay, Comparison

Journal: The Journal of clinical endocrinology and metabolism

Article Title: β2-Agonist Induces Net Leg Glucose Uptake and Free Fatty Acid Release at Rest but Not During Exercise in Young Men.

doi: 10.1210/jc.2018-01349

Figure Lengend Snippet: Figure 5. Muscle protein phosphorylation and representative Western blots for terbutaline (filled bars) and placebo (open bars) at rest and at cessation of knee extensor exercise. (a) Phosphorylation of PKA substrateSer/Thr. (b) Phosphorylation ratio of p-AktSer473/Akt total. (c) Representative blots. Values are means and upper bound of the 95% CI (n = 9 for rest; n = 8 for exercise). *P , 0.05, for treatment difference at sampling point. EX, cessation of exercise; R, rest.

Article Snippet: Primary antibodies used were p-PKA substrateSer/Thr (dilution, 1:500; no. 9621, Cell Signaling Technology, Leiden, Netherlands), Akt (dilution, 1:1000; no. 2967, Cell Signaling Technology), and p-AktSer473 (dilution, 1:1000; no. 9271, Cell Figure 1.

Techniques: Phospho-proteomics, Western Blot, Sampling

Journal: Oncotarget

Article Title: The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells

doi: 10.18632/oncotarget.28300

Figure Lengend Snippet: ( A – C ) Nanostring mRNA expression screening of (A) matriptase (ST14), (B) HAI-1 (SPINT1) and (C) HAI-2 (SPINT2) was performed on human myeloma cell lines ( n = 8). ( D , E ) Matriptase (D) overexpression and (E) knockdown was confirmed via Western blotting.

Article Snippet: A plasmid containing a shRNA specific for matriptase (sc-43911-V) and control plasmid (sc-108080) purchased from Santa Cruz Biotechnology was used to establish cells with stable knocked-down matriptase (RPMI-8226 shMatriptase and JJN-3 shMatriptase) and control cell lines (RPMI-8226 shMock and JJN-3 shMock), respectively.

Techniques: Expressing, Over Expression, Knockdown, Western Blot

Journal: Oncotarget

Article Title: The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells

doi: 10.18632/oncotarget.28300

Figure Lengend Snippet: Protein expression of HAI-1 and HAI-2 was determined using Western blotting. ( A ) INA-6 and U266 matriptase overexpression (Matriptase) and control (Mock) cells, and ( B ) RPMI-8226 and JJN-3 matriptase knockdown (shMatriptase) and control (shMock) cells. One representative of at least three independent experiments is shown.

Article Snippet: A plasmid containing a shRNA specific for matriptase (sc-43911-V) and control plasmid (sc-108080) purchased from Santa Cruz Biotechnology was used to establish cells with stable knocked-down matriptase (RPMI-8226 shMatriptase and JJN-3 shMatriptase) and control cell lines (RPMI-8226 shMock and JJN-3 shMock), respectively.

Techniques: Expressing, Western Blot, Over Expression, Control, Knockdown

Journal: Oncotarget

Article Title: The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells

doi: 10.18632/oncotarget.28300

Figure Lengend Snippet: Cell proliferation was measured in INA-6 Matriptase overexpression (Matriptase) and control (Mock) cells by the CellTiter-Glo assay. The mean (±SD) of four independent experiments is shown. Each dot represents one technical replicate and dots in the same color correspond to one of the biological replicates. Triangles represent the mean of each biological replicate. p -value was calculated by unpaired Student’s t -test based on the average from each independent experiment. * p ≤ 0.05.

Article Snippet: A plasmid containing a shRNA specific for matriptase (sc-43911-V) and control plasmid (sc-108080) purchased from Santa Cruz Biotechnology was used to establish cells with stable knocked-down matriptase (RPMI-8226 shMatriptase and JJN-3 shMatriptase) and control cell lines (RPMI-8226 shMock and JJN-3 shMock), respectively.

Techniques: Over Expression, Control, Glo Assay

Journal: Oncotarget

Article Title: The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells

doi: 10.18632/oncotarget.28300

Figure Lengend Snippet: ( A , B ) Migration of (A) INA-6 and (B) U266 matriptase overexpression (Matriptase) and control (Mock) cells. The promigratory cytokines HGF and/or SDF-1α were added to the lower wells as indicated. ( C ) Basal migration of matriptase knockdown (shMatriptase) and control cells (shMock) in RPMI-8226 and JJN-3. ( D ) Src activation in INA-6 Matriptase and Mock. Cells were starved for 6 h in serum-free medium. Subsequently, cells were stimulated with HGF and SDF-1α for 10 and 30 min and probed with antibodies as indicated. ( E ) Migration of INA-6 Matriptase and Mock cells. The combination of HGF and SDF-1α were added to the lower wells in all conditions. DMSO or PP2 Src inhibitor were added in concentrations as indicated to both upper and lower wells. (A–C and E) Cells were seeded in the upper well of a two-chamber transwell migration assay. After 24 h incubation, cells in the bottom wells were counted and the percentage of migrated cells calculated. Bars in (A), (B), (C) and (E) represent the mean (±SD) of at least two repeated counts in two independent measurements. Concentrations of HGF and SDF-1α was 150 ng/mL and 75 ng/mL, respectively, in all experiments. One representative of three independent experiments is shown in all figures. p -values were calculated by unpaired Student’s t -test. ns = not significant ( p > 0.05), * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: A plasmid containing a shRNA specific for matriptase (sc-43911-V) and control plasmid (sc-108080) purchased from Santa Cruz Biotechnology was used to establish cells with stable knocked-down matriptase (RPMI-8226 shMatriptase and JJN-3 shMatriptase) and control cell lines (RPMI-8226 shMock and JJN-3 shMock), respectively.

Techniques: Migration, Over Expression, Control, Knockdown, Activation Assay, Transwell Migration Assay, Incubation