constructs 64624  (Addgene inc)

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 1 (A) Graphical overview of the experimental set-up, starting with the isolation of seminal fluid extracellular vesicles (SF-EVs) from leftover semen samples via ultracentrifugation and size-exclusion chromatography. The purified SF-EV stocks showed distinctive TMPRSS2 enzyme activity and were characterised for size distribution via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Then, we confirmed surface-exposed TMPRSS2 on the SF-EVs via high-sensitivity flow cytometry, triggering us to develop an SF-EV based in vitro screening assay for TMPRSS2-inhibitors. (B) Architecture of TMPRSS2. Native TMPRSS2 constitutes the N-terminal cytoplasmic, the transmembrane, the LDL- receptor class A, the scavenger receptor cysteine-rich and the peptidase S1 domains. The recombinant TMPRSS2 (dasTMPRSS2, 44 kDa) constitutes the LDL-receptor class A, the scavenger receptor cysteine-rich, the peptidase S1 domain (26 kDa) and an AviTag and C-terminal 8xHIS-tag (not shown), with the enterokinase-cleavable sequence DDDDK255 at the red dot replacing the native SRQSR255 amino acid sequence. TM: transmembrane domain (amino acids 85–105); N and C: N- and C-terminus, respectively. Numbers indicate amino acid position according to UniProt O15393. Figure created with BioRender.com.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Isolation, Size-exclusion Chromatography, Purification, Activity Assay, Transmission Assay, Electron Microscopy, Flow Cytometry, In Vitro, Screening Assay, Recombinant, Sequencing

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 3 SF-EVs and recombinant TMPRSS2 both display specific trypsin-like enzyme activity. (A) Michaelis–Menten plot of initial reaction velocities (V0) for kinetic parameter estimation of the generic Boc-QAR-AMC fluorogenic substrate cleaved by SF-EVs (± 3.3 × 1010 vesicles/mL), after curve fitting in GraphPad (Supporting Information Equation 1). Michaelis constant (Km: 278 ± 52 µM) is indicated by dotted line. (B) Plot of the fractional enzyme activity in SF-EVs (3.3 × 1010 vesicles/mL) versus [Nafamostat mesylate] by measuring the initial reaction velocities at 37◦C in the presence of 100 µM Boc-QAR-AMC fluorogenic substrate and curve fitting for absolute IC50 determination in GraphPad (Supporting Information Equation 2). IC50 (31 ± 5 pM) is indicated by dotted line. (C) Michaelis–Menten plot of initial reaction velocities for kinetic parameter estimation of the generic Boc-QAR- AMC fluorogenic substrate cleaved by recombinant TMPRSS2 (50 pM), after curve fitting in GraphPad (Supporting Information Equation 1). Michaelis constant (Km: 133 ± 16 µM) is indicated by dotted line. (D) Plot of the fractional enzyme activity of recombinant TMPRSS2 (50 pM) versus [Nafamostat mesylate] by measuring the initial reaction velocities at 37◦C in the presence of 100 µM Boc-QAR-AMC fluorogenic substrate and curve fit for absolute IC50 in GraphPad (Supporting Information Equation 2). IC50 (68 ± 3 pM) is indicated by dotted line. All data are shown as mean ± s.d. and were all performed in biological triplicate (n = 3). Graphs were created with GraphPad Prism v9.1.1.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Recombinant, Activity Assay

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 4 High-sensitivity flow cytometry analysis of SF-EVs. Density plots (R670/30-A vs. SP SSC-H) of purified SF-EVs co-stained with CFDA- SE and (A) anti-human TMPRSS2-APC or (B) anti-human CD26-APC, followed by bottom-up density gradient ultracentrifugation. The plots show a 60 s analysis of fraction 6 (1:50 dilution in PBS) and are representative of technical duplicate or triplicate (n = 2–3). All axes are denoted in arbitrary units. Gates were set as described in the MIFlowCyt checklist (Table S5). (A) The density plot of CFDA-SE positive events labelled with anti-human TMPRSS2 antibody demonstrates a moderate increase in the APC signal (R670/30-A). (B) The density plot of CFDA-SE positive events labelled with anti-human CD26 antibody demonstrates a low increase in the APC signal (R670/30-A).

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Flow Cytometry, Purification, Staining

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 5 Detection of TMPRSS2 in the seminal fluid-derived extracellular vesicle preparations. The detected TMPRSS2 band perfectly matches the theoretical weight of the Peptidase S1 domain (26 kDa) as calculated with ExPASy ProtParam, with the less prominent band probably arising due to off-target auto-activation of the protein (Gasteiger et al. 2005). The band detected in the PC3 cells most closely correlates to the extracellular topological domain of TMPRSS2 and can also slightly be detected in the SF-EVs. A molecular weight ladder (Std) was loaded on each gel. Std, PageRuler Plus Prestained Protein Ladder; EV, seminal-fluid extracellular vesicles; PC3, prostate cancer cell lysate.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Derivative Assay, Activation Assay, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: p38 mitogen-activated protein kinase (MAPK) contributed to kainic acid-induced seizure in WT mice but not Kv4.2TA mice. ( A ) Time course of mean behavioral seizure score following kainic acid injection. The mean behavioral seizure score was significantly reduced in Kv4.2TA mice compared to WT mice. Furthermore, p38 inhibitor SB 203580 significantly reduced behavioral seizure score following kainic acid injection in WT mice but not in Kv4.2TA mice, n = 13–15 for each group, two-way ANOVA, * p < 0.05. ( B ) Total behavioral seizure score for each group, n = 13–15 for each group, t -test, * p < 0.05. ( C ) Latency to stage 3 seizure for each group. n = 13–15 for each group, t -test, ** p < 0.01.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: p38 MAPK contributes to kainic acid-induced Kv4.2 phosphorylation at T607. ( A ) SB 203580, a potent p38 inhibitor (20 mg/kg, i.p., 15 min), blocked kainic acid-induced phosphorylation of Kv4.2 T607 in mouse hippocampus. ( B ) Statistical analysis of the effect of SB 203580 on kainic acid-induced phosphorylation of Kv4.2 at Thr607 in mouse hippocampus, n = 4–6 in each group, t -test, ** p < 0.01.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: p38 MAPK colocalizes with Kv4.2. ( A ) HEK293T cells were transfected with GFP-Kv4.2 and Flag-p38. Cells were fixed and stained with GFP and Flag to show co-localization. Scale bar: 20 μm. ( B ) High magnification images and line scan analysis of colocalization. Scale bar: 5 μm. ( C ) Mouse brains were co-stained with Kv4.2 and pp38 antibody. Phosphorylated p38 is localized in the cell body and dendrites as well. Scale bar: 20 μm. ( D ) High magnification images showing Kv4.2 and pp38 colocalized in dendrites, as indicated with arrow heads. Scale bar: 5 μm.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Transfection, Staining

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: Kainic acid activates p38 MAPK in both WT and Kv4.2TA mice. ( A ) Immunostaining analysis showed p38 phosphorylation increased with kainic acid administration (25 mg/kg, i.p., 30 min) in mouse hippocampus, n = 26 cells in each group, t -test, *** p < 0.001. ( B ) Western blot analysis showed p38 phosphorylation increased with kainic acid administration (25 mg/kg, i.p., 30 min) in hippocampus in both WT and Kv4.2TA mice, n = 4–6 cells in each group, t -test, *** p < 0.001.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Immunostaining, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: p38 impacts hippocampal pyramidal neuron excitability through Kv4.2. ( A ) Current step of +300 pA induces repetitive firing in pyramidal neurons recorded from WT and Kv4.2TA mice with or without SB 203580 treatment. Scale 40 mV/250 ms. Square current inset 300 pA. ( B ) Sequential somatic current injections increasing in magnitude reveal p38 kinase inhibition reduces AP firing frequency in WT hippocampal neurons at +300 pA relative to vehicle ( n = 15 in vehicle, n = 19 in treatment; two-way ANOVA, * p < 0.05). Kv4.2TA neurons display reduced firing frequency at +300 pA relative to WT in vehicle, which is augmented in the presence of SB 203580 such that current magnitudes of +200 and +250 pA also exhibit significant differences ( n = 18 in vehicle, n = 14 in SB 203580; two-way ANOVA, * p < 0.05; *** p < 0.001). ( C ) Inter-spike intervals measured between the first two spikes in a train evoked by 150 pA injection display no significant difference among groups. Kruskal–Wallis test, p > 0.05. ( D ) Ramp current injections evoke repetitive firing in all pyramidal neurons recorded in each condition. Arrow indicates point at which action potential (AP) threshold, rheobase, and latency to fire were measured. Ramp current inset 400 pA/s. ( E ) Minimum current to elicit AP firing at threshold (rheobase) is not significantly different among the populations. One-way ANOVA, p > 0.05. ( F ) Latency to fire in response to ramp injection is not significantly different among populations. Kruskal–Wallis test, p > 0.05.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Inhibition, Injection