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: Molecular Cancer

Article Title: Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

doi: 10.1186/1476-4598-5-69

Figure Lengend Snippet: HEK293 cells transfected with MT1-MMP cDNA invade 3D collagen gels in response to LPA . (A) Tumor cell lysates were prepared for Western blot analysis. Lysates were probed for MT1-MMP to assess protein expression in the four tumor cell lines. Lysates were probed for Actin as a loading control. (B) HEK293 cells were transfected with the pAdTrack-CMV plasmid as a control, or plasmids encoding MT1-MMP, MT2-MMP, or MT3-MMP cDNA 24 hours prior to placement in invasion assays. Cells were allowed to invade 2.0 mg/ml collagen gels in the presence or absence of 1 μM LPA. Data are expressed as mean numbers of invading cells per HPF (20×) (± S.D.) from a minimum of 20 fields. (C) Lysates from HEK293 cells transfected with cDNAs encoding the designated genes were prepared for Western blot analysis and probed for GFP, MT1-MMP, MT2-MMP, MT3-MMP, or Actin as a loading control. TRK = pAdTrack-CMV, MT1 = MT1-MMP, MT2 = MT2-MMP, MT3 = MT3-MMP.

Article Snippet: Human MT1-MMP (AF918, R&D Systems, Minneapolis, MN), MT3-MMP (RP1-MMP-16, Triple Point Biologics, Forest Grove, OR), and Rac1 (ARC01, Cytoskeleton, Denver, CO), and monoclonal antibodies directed against human MT2-MMP (MAB916, R&D Systems), Lamin A/C (MAB3211, Chemicon Corp), Actin (JLA-20, Calbiochem, San Diego, CA), RhoA (ARH01, Cytoskeleton), and Cdc42 (610929, BD Tansduction, San Jose, CA) were used for Western blot analysis as described previously [ ]. si GENOME SMART pool ® reagents were products of Dharmacon (Lafayette, CO).

Techniques: Transfection, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Size exclusion chromatography (SEC) curves exhibiting the formation of a complex between SENP3 protease domain 302 to 574aa and PELP1 SLiM -containing peptide 761 to 796aa. SDS-PAGE and total protein staining of SEC fractions are displayed below the x axis. ( B ) AlphaFold3 structural models of proSUMO substrates used in endopeptidase cleavage assays. Sequences of the proSUMO C-terminal tails (cleavage sites labeled with arrows) are shown below the structural models. ( C ) SDS-PAGE and total protein stain of in vitro SENP3 ± MBP-PELP1 SLiM endopeptidase activity assays against proSUMO1, −2, and −3 substrates. Decreasing concentrations (2500 to 0.12 nM) of SENP3 enzyme ± MBP-PELP1 761 to 796aa was incubated for 1 hour at 37°C with 5 μM proSUMO substrate. Cleaved proSUMO product is labeled with asterisks. Quantification curves representing percent (%) proSUMO endopeptidase cleavage correspond to the gel images and the enzyme concentration range was 1000 to 15.63 nM. SD was calculated from three independent experiments ( n = 3). ( D ) SDS-PAGE and total protein stain of in vitro SENP3 (C532A) ± MBP-PELP1 SLiM endopeptidase activity assay, illustrating no activity. ( E ) Quantification curves representing percent (%) proSUMO2 endopeptidase cleavage by SENP protease domain + PELP1 SLiM , SENP3 protease domain alone, or SENP5 protease domain alone during a time course (0 to 450 s). Enzyme concentration was kept constant at 1000 nM along with substrate concentration at 5 μM. Percent cleavage was calculated as in (C). SD was calculated from three independent assay samples ( n = 3). Raw gel images are displayed in fig. S13A. ( F ) Differential scanning fluorimetry curves exhibiting the thermal stabilization of the SENP3 protease domain upon addition of short (“s,” amino acids 764 to 781) and long (“l,” amino acids 764 to 792) PELP1 SLIM peptides. Boltzmann and first derivative curves are shown on the top and bottom of the panel, respectively. Values for change in T m is only shown for the first derivative.

Article Snippet: N-terminal 6× His-tagged human SENP3 protease domain (302 to 574aa, E. coli codon-optimized gene synthesized by Genscript in pET11a vector) and SENP5 protease domain [567 to 755aa, plasmid no. 16358 ( ) from Addgene in pET28a vector] were used for in vitro binding and deSUMOylation assays.

Techniques: Size-exclusion Chromatography, SDS Page, Staining, Labeling, In Vitro, Activity Assay, Incubation, Concentration Assay