Journal: American Journal of Translational Research

Article Title: PIAS3/SOCS1-STAT3 axis responses to oxidative stress in hepatocellular cancer cells

doi:

Figure Lengend Snippet: Effect of H2O2 treatment on oxidative stress induction and malignant processes in HCC cells. A. H2O2 (50 μM for 24 h) and/or antioxidant TA treatment. Total antioxidant capacity (TAC) in HepG2 cells was determined. VCEAC: vitamin C equivalent antioxidant capacity (n=3). B. Protein carbonyl content in HepG2 cells was detected (n=3). C. Western blotting detected the expression of Nrf2 protein in cells (n=3). The band density for Nrf2 protein are shown (n=3). D. HepG2 cell proliferation 48 h after different treatments (n=3). E. Cell growth of HepG2 cells 24-72 h post treatment (n=3). F. Cell apoptosis in HepG2 cells. G. Invasion ability of HepG2 cells (n=3). Scale bar, 50 μm. H. Migration capacity of HepG2 cells (n=3). Scale bar, 50 μm. *P<0.05, **P<0.01.

Article Snippet: Immunohistochemical staining Immunohistochemical staining was performed according to the manufacturer’s instructions with the following reagents and instruments: horse serum (RTU Vectastain Kit, PK-7200), 1:200 mouse anti-Nrf2 antibody (AF3925, R&D Systems), ABC reagent (LS-J1026-1, Vector labs), and IHC slide staining system (NanoMtrx 100, BioGenex).

Techniques: Western Blot, Expressing, Migration

Journal: American Journal of Translational Research

Article Title: PIAS3/SOCS1-STAT3 axis responses to oxidative stress in hepatocellular cancer cells

doi:

Figure Lengend Snippet: Effects of PIAS3 and SOCS1 overexpression and colivelin treatment on oxidative stress induction and malignant processes of HCC cells. HepG2 cells were transfected with PIAS3 or SOCS1 overexpression vector for 36 h and then treated with H2O2 (50 μM) and/or 0.5 μM CO for 24 h. A. Total antioxidant capacity (TAC) in HepG2 cells was determined. VCEAC: vitamin C equivalent antioxidant capacity (n=3). B. Protein carbonyl content in HepG2 cells was detected (n=3). C. Western blotting detected the expression of Nrf2 protein in cells (n=3). Quantification data from three independent repeats were showed below the blot. D. HepG2 cell proliferation 48 h after different treatments (n=3). E. MTT assays detected cell growth of HepG2 cells 24-72 h post treatment (n=3). F. Cell apoptosis of HepG2 cells. G. Invasion ability of HepG2 cells (n=3). Scale bar, 50 μm. H. Migration capacity of HepG2 cells (n=3). Scale bar, 50 μm. *P<0.05, **P<0.01.

Article Snippet: Immunohistochemical staining Immunohistochemical staining was performed according to the manufacturer’s instructions with the following reagents and instruments: horse serum (RTU Vectastain Kit, PK-7200), 1:200 mouse anti-Nrf2 antibody (AF3925, R&D Systems), ABC reagent (LS-J1026-1, Vector labs), and IHC slide staining system (NanoMtrx 100, BioGenex).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Expressing, Migration

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 stabilizes Nrf2 through binding with Keap1. A HEK293 cells transfected with Flag-TRIM15, HA-Keap1, and Myc-Nrf2 were subjected to immunoprecipitation with HA antibody. Lysates were analyzed by western blotting. B TRIM15 reduced the interaction between Nrf2 and Keap1. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. C – F Subcellular fractionation was used to isolate cytoplasmic and nuclear proteins, and immunoblotting was performed to examine the localization of Nrf2 following the downregulation or overexpression of TRIM15. Nuclear and cytoplasmic levels of Nrf2 are quantified. G Effect of TRIM15 knockdown (H1299 cells) or overexpression (H1650 cells) on the mRNA expression of the Nrf2-regulated genes. NAD(P)H quinone dehydrogenase1(NOQ1), thioredoxin (TXN), peroxiredoxin 1(PRDX1), hemoxygenase 1(HMOX1), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase μ1(GSTM1), glutathione S-transferase μ3(GSTM3), ferritin light chain (FTL). H Representative IHC staining images of Nrf2 in the same set of NSCLC tissue slices. Correlation analysis of TRIM15 and Nrf2 expression in NSCLC samples. Spearman correlation coefficients are shown. Scale bars, 100 μm. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Fractionation, Over Expression, Knockdown, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15-mediated Nrf2 signaling regulates growth and invasion in NSCLC cells in vitro. A Western blot analyses of TRIM15, Nrf2, Keap1, and Nrf2 target NQO1 in H1299 cells with TRIM15 knockdown with or without subsequent Nrf2 overexpression and H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. B ARE Luc reporter activity assessed in H1299 cells expressing shTRIM15, sh TRIM15 + Nrf2 or H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. Up-regulating of Nrf2 expression in H1299 cells or down-regulating of Nrf2 expression in H1650 cells was set as a control. C – F Cell proliferation ( C , D ), invasion ( E ) and ROS formation ( F ) in H1299 cells with or without shTRIM15 and Nrf2 rescue or in H1650 cells with or without TRIM15 overexpression and shNrf2 rescue. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vitro, Western Blot, Knockdown, Over Expression, Activity Assay, Expressing, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 mediated increase in Nrf2 regulates growth and invasion in vivo. A Nude mice were randomized into three groups and subcutaneously injected with H1650 cells that had been transfected with control (empty vector), TRIM15, or TRIM15 + shNrf2 plasmids. Tumors formed in nude mice were collected 30 days after grafting, and the tumor weight were measured. B Measurement of tumor volume in experimental groups over time. C Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in harvested tumors. D , E Up-regulation of TRIM15 significantly promoted lung metastasis in H1650 xenograft nude mice models, whereas the suppression of Nrf2 prevented the tumor metastasis of TRIM15 overexpressing cells. Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. F , G A representative image of tumor growth in nude mice subcutaneously inoculated with H1299 cells tranfected with shCtrl, shTRIM15 or shTRIM15 + Nrf2 plasmids. Tumor volumes were measured on the indicated days. H Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in xenograft tumors. I Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. J TRIM15 was significantly upregulated in NSCLC and that increased TRIM15 was associated with poor survival. TRIM15 promoted tumor proliferation and metastasis by activating Nrf2 signaling. Furthermore, TRIM15 regulated Nrf2 activity by modulating Keap1 and inducing its ubiquitination and degradation in NSCLC cells. Activation of Nrf2 facilitated tumor cell proliferation and invasion. N.S. represents no significant. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vivo, Injection, Transfection, Control, Plasmid Preparation, Western Blot, Staining, Activity Assay, Activation Assay, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: Self-immolative nanocapsules precisely regulate depressive neuronal microenvironment for synergistic antidepression therapy

doi: 10.1186/s12951-023-02008-9

Figure Lengend Snippet: In vivo antidepressive effects of nanocapsules. A In vivo Cy7.5 fluorescence of C57BL/6J mice and CUMS mice after intravenous injection with VCNCs-Cy7.5, CNCs-Cy7.5, and VNCs-Cy7.5 at 400 μg 5-HT/kg for 3 h and 7 h. Living Cy 5.5 fluorescence images of mice after treatment with VCNCs-Cy5.5, CNCs-Cy5.5, and VNCs -Cy5.5 (C 5-HT : 400 μg /kg) for 3 h. The representative western blot analysis of B Nrf 2 in the hippocampus of brain across all groups. The other two replicates were presented in Additional file : Fig. S38. Reverse transcription quantitative polymerase chain reaction analysis (RT-qPCR) of relative mRNA levels of C NLRP3 in the hippocampi of mice in all groups (n = 3). The levels of hippocampal D IL-6 in the mice hippocampi after treatment were detected using ELISA kits. The results were normalized by the protein concentration of each sample (n = 3). E ROS/DAPI staining and F GFAP / DAPI staining brains in groups subjected to different treatments. Analysis of hippocampal BDNF expression using G western blot, H RT-qPCR, I ELISA kits and J immunohistochemistry slides. The significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05, **P < 0.01, *** P < 0.001

Article Snippet: The sample solutions were then measured using IL-6 (Elabscience, #E-MSEL-M0001, China), IL-1β (Elabscience, #E-EL-M0037c, China), TNF-α (Elabscience, #E-EL-M3063, China), BDNF (Elabscience, #E-EL-M0203c, China), Nrf2 (Cusabio, #CSB-E16188m, China), and 5-HT ELISA kits (Elabscience, #E-EL-0033c China) according to the manufacturer’s instructions.

Techniques: In Vivo, Fluorescence, Injection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Protein Concentration, Staining, Expressing, Immunohistochemistry

Journal: Journal of Nanobiotechnology

Article Title: Self-immolative nanocapsules precisely regulate depressive neuronal microenvironment for synergistic antidepression therapy

doi: 10.1186/s12951-023-02008-9

Figure Lengend Snippet: Effects of nanocapsules on in vivo 5-HT level and neuroprotection. A 5-HT IHC and B nissil and HE staining of brains after administering with fluoxetine, VCNCs, CNCs, and VNCs. C The levels of hippocampal 5-HT in the mice hippocampi after treatment were detected using ELISA kits. The results were normalized by the protein concentration of each sample (n = 3). The significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05. D The in vivo therapeutic outcomes of nanocapsules

Article Snippet: The sample solutions were then measured using IL-6 (Elabscience, #E-MSEL-M0001, China), IL-1β (Elabscience, #E-EL-M0037c, China), TNF-α (Elabscience, #E-EL-M3063, China), BDNF (Elabscience, #E-EL-M0203c, China), Nrf2 (Cusabio, #CSB-E16188m, China), and 5-HT ELISA kits (Elabscience, #E-EL-0033c China) according to the manufacturer’s instructions.

Techniques: In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Life

Article Title: Modulation of Oxidative and ER Stress Pathways by the ADAM17 Inhibitor GW280264X in LPS-Induced Acute Liver Injury

doi: 10.3390/life15121877

Figure Lengend Snippet: Effects of GW280264X on redox regulatory proteins in liver tissue. ( A ) Kelch-like ECH-associated protein 1 (Keap1), ( B ) nuclear factor erythroid 2 2-related factor 2 (Nrf2), and ( C ) glutathione (GSH) levels. Data are presented as mean ± SEM ( n = 5). * p < 0.05 vs. 0.9% NaCl, # p < 0.05 vs. LPS. Black squares represent individual data points for each animal.

Article Snippet: Commercial ELISA kits were used to quantify liver tissue levels of KEAP1 (Cat. No. E2198Mo, BT-Laboratory, Shanghai, China), CHOP (Cat. No. E2718Mo, BT-Laboratory), 4-HNE (Cat. No. E1293Mo, BT-Laboratory), MDA (Cat. No. E0625Mo, BT-Laboratory), NRF2 (Cat. No. E1367Mo, BT-Laboratory), TNF-α (Cat. No. E-EL-M3063, Elabscience, Houston, TX, USA), GRP78 (Cat. No. E-EL-M2696, Elabscience, Houston, TX, USA), glutathione (GSH) colorimetric assay kit (Cat No. E-EL-0026, Elabscience, Houston, TX, USA) and ATF6 (Cat. No. ELK4479, Elk Biotechnology, Wuhan, China).

Techniques:

Journal: Journal of Dental Research

Article Title: Autophagy Plays a Crucial Role in Ameloblast Differentiation

doi: 10.1177/00220345231169220

Figure Lengend Snippet: Identification of genes with compromised expression in the teeth of autophagy-deficient mice. ( A ) Schematic diagram of binding sites (BSs) for NRF2 in the promoter region (10 kb upstream from transcription start site) of each gene related to amelogenesis imperfecta. The conserved NRF2 BSs among 8 species were selected for experimental validation. ( B ) Quantitative reverse transcription polymerase chain reaction analyses for the indicated genes of upper incisors from wild-type (blue bars) and Atg7 cKO (green bars) mice. *** P < 0.001. n = 6 per group. ( C ) Chromatin immunoprecipitation analyses for each BS in Dlx3 , Klk4 , Nectin1 , Bcl11c , Pax9 , and Ltbp3 . *** P < 0.001; ns, not significant. n = 6 per group.

Article Snippet: Either the mouse full-length Nrf2/Nfe2l2 cDNA vector (MG56971-U; Sino Biological, Inc.) or the pUC19 backbone vector (50005; addgene) was transfected with Lipofectamine 3000 (Thermo Fisher Scientific) into WT mHAT9d cells, according to the manufacturer’s protocol; 24 h after transfection, the cells were cultured with ameloblast differentiation medium (including 15 µg/mL retinoic acid [R2625, Sigma Aldrich] and 0.1 μM dexamethasone [D4902, Sigma Aldrich]) for 48 h in order to induce ameloblast differentiation.

Techniques: Expressing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Journal of Dental Research

Article Title: Autophagy Plays a Crucial Role in Ameloblast Differentiation

doi: 10.1177/00220345231169220

Figure Lengend Snippet: NRF2-dependent regulation of genes in autophagy-deficient ameloblasts. ( A ) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analyses for the indicated genes in mHat9d cells treated with a control (blue bars) and Nrf2 overexpression (red bars) vector. *** P < 0.001. n = 6 per group. ( B ) Immunoblotting for the indicated molecules in wild-type (WT), Atg7 knockout (KO), and Atg3 KO mHAT9d cells. ( C ) Transmission electron microscopy in WT, Atg7 KO, and Atg3 KO mHAT9d cells. Yellow arrows indicate the autophagosomes. Red arrow indicates vesicle-like abnormal structures. Scale bars: 2 μm. ( D ) Quantitative RT-PCR analyses for the indicated genes in WT and Atg7 KO mHAT9d cells (upper panel) and WT and Atg3 KO mHAT9d cells (lower panel) with and without Nrf2 knockdown (KD). ** P < 0.005. *** P < 0.001. n = 6 per group.

Article Snippet: Either the mouse full-length Nrf2/Nfe2l2 cDNA vector (MG56971-U; Sino Biological, Inc.) or the pUC19 backbone vector (50005; addgene) was transfected with Lipofectamine 3000 (Thermo Fisher Scientific) into WT mHAT9d cells, according to the manufacturer’s protocol; 24 h after transfection, the cells were cultured with ameloblast differentiation medium (including 15 µg/mL retinoic acid [R2625, Sigma Aldrich] and 0.1 μM dexamethasone [D4902, Sigma Aldrich]) for 48 h in order to induce ameloblast differentiation.

Techniques: Reverse Transcription Polymerase Chain Reaction, Over Expression, Plasmid Preparation, Western Blot, Knock-Out, Transmission Assay, Electron Microscopy, Quantitative RT-PCR

Journal: Aging Cell

Article Title: Parkinson's disease, aging and adult neurogenesis: Wnt/β‐catenin signalling as the key to unlock the mystery of endogenous brain repair

doi: 10.1111/acel.13101

Figure Lengend Snippet: Interventions indirectly targeting WβC‐signalling activation (WβC‐AC)‐targeted in the central nervous system

Article Snippet: Accordingly, several lines of evidence including in vivo Nrf2‐deficient mouse models, post‐mortem studies of PD brains, and genetic association studies of patients indicate a link between Nrf2 dysregulation and PD pathogenesis (Johnson & Johnson, ).

Techniques: Activation Assay, In Vitro, Expressing, In Vivo, Inhibition, Activity Assay, Optogenetics, Microscopy

Journal: Aging Cell

Article Title: Parkinson's disease, aging and adult neurogenesis: Wnt/β‐catenin signalling as the key to unlock the mystery of endogenous brain repair

doi: 10.1111/acel.13101

Figure Lengend Snippet: Cross talk dialogue between inflammatory and WβC‐signalling pathways in MPTP‐induced SVZ plasticity is lost with age. A simplified scheme summarizing MPTP‐induced neuroinflammation and SVZ plasticity in young and aged mice via modulation of WβC‐signalling ( “Wnt on; Wnt off” ) is shown. In young mice, during the degeneration phase, hyperactivated M1 microglia contributes to the impairment of SVZ neurogenesis at different levels. By increasing oxidative and nitrosative stress and in synergy with MPTP/MPP + direct toxicity, microglial‐derived mediators (PHOX‐derived ROS, iNOS‐derived NO, and peroxynitrite) may act as molecular switch for cell signalling pathways critically involved in the physiological control of NSC homeostasis, with harmful consequences for astrocyte and NSC physiology, at least in part through GSK‐3 β activation, followed by phosphorylation and consequent degradation of β‐catenin. In young mice, after the acute inflammatory and degenerative phase, a regulatory circuit linking microglial activation and proinflammatory cytokine to Nrf2‐ARE protective pathway in SVZ, provides an efficient self‐adaptive mechanism against inflammatory/neurotoxin‐induced oxidative stress, switching the M1 microglial harmful phenotype, thus mitigating inflammation with a return to pre‐MPTP conditions. By contrast, the aging process, in synergy with MPTP exposure, negatively impacts on astrocytic Nrf2‐driven Hmox1 response within the SVZ niche in vivo. Hence, this process, resulting from an age‐dependent dysregulation of astrocyte‐microglia interactions, contributes to the exacerbated oxidative and inflammatory SVZ status and the decline of astrocyte Wnt‐dependent regulation, finally leading to NSC neurogenic impairment and loss of SVZ plasticity The mutual role of astrocyte–microglial interactions in the plasticity of SVZ response to MPTP is exemplified by the astrocyte's ability to overcome microglial inhibitory effects, also via cross talk with Wnt/β‐catenin signalling. Pharmacological mitigation of inflammation and oxidative stress or GSK‐β antagonism upregulates β‐catenin and successfully rescues NSC proliferation and neuroblast formation, a process associated with striatal DAergic neuroprotection, with further positive modulation of SVZ proliferation via D2 receptor (D2R) activated mechanisms

Article Snippet: Accordingly, several lines of evidence including in vivo Nrf2‐deficient mouse models, post‐mortem studies of PD brains, and genetic association studies of patients indicate a link between Nrf2 dysregulation and PD pathogenesis (Johnson & Johnson, ).

Techniques: Derivative Assay, Activation Assay, In Vivo

Journal: Aging Cell

Article Title: Parkinson's disease, aging and adult neurogenesis: Wnt/β‐catenin signalling as the key to unlock the mystery of endogenous brain repair

doi: 10.1111/acel.13101

Figure Lengend Snippet: Interventions directly targeting WβC‐signalling activation (WβC‐AC)‐targeted in the central nervous system

Article Snippet: Accordingly, several lines of evidence including in vivo Nrf2‐deficient mouse models, post‐mortem studies of PD brains, and genetic association studies of patients indicate a link between Nrf2 dysregulation and PD pathogenesis (Johnson & Johnson, ).

Techniques: Activation Assay, Expressing, Transduction, In Vivo, Binding Assay, Activity Assay, Transgenic Assay, Inhibition, Purification, In Vitro, Derivative Assay, Transplantation Assay, Functional Assay, Injection, Migration

Journal: Aging Cell

Article Title: Parkinson's disease, aging and adult neurogenesis: Wnt/β‐catenin signalling as the key to unlock the mystery of endogenous brain repair

doi: 10.1111/acel.13101

Figure Lengend Snippet: WβC‐signalling activation as a disease modifying strategy for PD. Schematic illustration of potential interventions targeting either indirectly or directly WβC‐signalling activation (WβC‐AC) in the central nervous system. As shown in the diagram WβC‐AC can be targeted at different steps (for details see the text and Tables and ). Indirect WβC‐AC, in blue, include several physical conditions/manipulations/pharmacological treatments, such as environmental enrichment, physical exercise, optogenetic and neural stimulation, or stem cell therapies, the administration of various classes of molecules, including anti‐oxidant and anti‐infammatory molecules (NO‐NSAIDs), Nrf2‐ and Nurr1‐agonists or herbal derivatives (curcumin, andrographolide, resveratrol). Direct activation of canonical Wnt/Fzd/β‐catenin cascade with: Fzd‐LRP5/6 Wnts surrogates ; SVZ‐NSC‐derived astrocyte's Wnt1 , in blue; small Wnt activators targeting Axin‐LRP6 (HLY78, SKL2001), PP2A agonists, in red (IQ1, Sodium selenite), directly interacting with multiple components of β‐catenin destruction complex; GSK‐3β antagonists, in red (VPA, LiCl, AR, BIO, CHIR, SB431542); or endogenous antagonists such as secreted FZD‐related proteins, sFRPs (WAY310666), in red. bFGF, basic fibroblast growth factor; casein kinase 1α (CK1α); Dishevelled, Dvl; Fzd, frizzled; GSK‐3β, glycogen synthase kinase 3β;NO‐NSAID, nitric oxide‐releasing non‐steroidal anti‐inflammatory drugs; Nrf2, nuclear factor erythroid 2‐related factor 2; NSC, neural stem/progenitor cell; Nurr1, nuclear receptor related 1 protein; sFRP, secreted Frizzled‐related proteins; SVZ, sub‐ventricular zone; tumor suppressor adenomatous polyposis coli, APC; WβC‐AC, Wnt/β‐catenin signalling activation

Article Snippet: Accordingly, several lines of evidence including in vivo Nrf2‐deficient mouse models, post‐mortem studies of PD brains, and genetic association studies of patients indicate a link between Nrf2 dysregulation and PD pathogenesis (Johnson & Johnson, ).

Techniques: Activation Assay, Derivative Assay