Journal: International Journal of Biological Sciences

Article Title: NUDT7 Modulates the UBA52-SREBF1 Signaling Axis to Promote PRRSV Replication via Lipid Synthesis

doi: 10.7150/ijbs.127844

Figure Lengend Snippet: The transcription factor ETS1 regulates NUDT7 expression in PRRSV-infected cells. The expression of NUDT7 mRNA and protein in PAM cells (A-B) and Marc-145 cells (C-D) was assessed using RT-qPCR and Western blotting at intervals from 0 to 36 hours following PRRSV infection. (E-F) NUDT7 mRNA and protein levels in PAM cells were assessed 24 hours after PRRSV infection at MOIs of 0, 0.5, 1, and 1.5 using RT-qPCR and Western blotting. Western blot data were semi-quantified and normalized using β-actin as the loading control. (G) Immunohistochemical analysis of NUDT7 and PRRSV-Nsp2 expression in negative control and PRRSV-infected cells (left panel). Scale bar: 25 μm. The right panel presents quantitative data on the relative fluorescence intensity of NUDT7. (H) Analysis of luciferase activity in PAM cells after transfection with pGL3-Basic luciferase vector carrying a truncated NUDT7 promoter construct (-2100 to -1). (I) JASPAR-predicted transcription factor binding sites on the NUDT7 promoter. (J) Relative mRNA levels of predicted genes in PAM cells 24 hours post-infection (hpi) with PRRSV (assessed by RT-qPCR). (K) PAM cells infected with PRRSV at MOI=1 or mock-infected. Cells were harvested at specified time points. ETS1 expression was analyzed by Western blotting. ACTB served as the loading control. (L) JASPAR-generated ETS1 DNA motif sequence logo. (M) PAM cells co-transfected with designated reporter plasmids and phRL-TK; cells harvested after 48 hours for dual luciferase assay. (N-O) NUDT7 relative levels in the ETS1 overexpressed PAM cells explored using western blotting and RT-qPCR.

Article Snippet: HEK293T (ATCC, CRL-11268) and Marc-145 (ATCC, CRL-12231) cells were maintained in DMEM (Gibco, C11995500BT) with 10% FBS (PAN-Biotech, P30-1406). iPAMs, an immortalized porcine alveolar macrophage cell line expressing the PRRSV receptor CD163, were cultured in RPMI 1640 medium (Gibco, catalog no.).C22400500BT) with 10% fetal bovine serum (FBS) added.

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Control, Immunohistochemical staining, Negative Control, Fluorescence, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct, Binding Assay, Generated, Sequencing

Journal: International Journal of Biological Sciences

Article Title: NUDT7 Modulates the UBA52-SREBF1 Signaling Axis to Promote PRRSV Replication via Lipid Synthesis

doi: 10.7150/ijbs.127844

Figure Lengend Snippet: PRRSV Nsp2 interacts with NUDT7. (A) Expression vectors encoding PRRSV proteins were transfected into Marc-145 cells. NUDT7 levels were assessed using luciferase assays and RT-qPCR. (B) NUDT7 levels were assessed via western blotting 36 hours after transfection with expression vectors for PRRSV-encoded proteins. (C) An exogenous co-immunoprecipitation (Co-IP) assay was conducted in HEK293T cells transfected with Myc-Nsp2 and either Flag-NUDT7 or an empty vector. Following transfection, cell lysates underwent immunoprecipitation with a Flag antibody and subsequent immunoblotting with both Myc and Flag antibodies. (D) Marc-145 cells were transfected with MYC-NSP2 and FLAG-NUDT7, then subjected to immunofluorescence staining using anti-FLAG (green), anti-Myc (red), and Hoechst 33258 (blue). (E) Co-IP experiments showing the interaction of Flag-Nsp2 with endogenous NUDT7 in iPAM cells. (F) The schematic diagram of Myc-Nsp2 deletion mutants. (G) HEK293T cells were transfected for 36 hours with plasmids encoding Flag-NUDT7 and either Myc-Nsp2 or specified Myc-Nsp2 deletion mutants. Subsequent co-immunoprecipitation was performed using anti-Flag beads, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Triangle labeled Myc-Nsp2 or specified Myc-Nsp2 deletion mutants. (H) Truncated Nsp2 and NUDT7 colocalization (Left). Pearson's correlation coefficient (Right) was used to express co-localization analysis, measured for individual cells using Image J. * p < 0.05 ; ** p < 0.01 ; *** p < 0.001 ; **** p < 0.0001 .

Article Snippet: HEK293T (ATCC, CRL-11268) and Marc-145 (ATCC, CRL-12231) cells were maintained in DMEM (Gibco, C11995500BT) with 10% FBS (PAN-Biotech, P30-1406). iPAMs, an immortalized porcine alveolar macrophage cell line expressing the PRRSV receptor CD163, were cultured in RPMI 1640 medium (Gibco, catalog no.).C22400500BT) with 10% fetal bovine serum (FBS) added.

Techniques: Expressing, Transfection, Luciferase, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Staining, Labeling

Journal: International Journal of Biological Sciences

Article Title: NUDT7 Modulates the UBA52-SREBF1 Signaling Axis to Promote PRRSV Replication via Lipid Synthesis

doi: 10.7150/ijbs.127844

Figure Lengend Snippet: PRRSV infection was promoted by NUDT7. (A-B) Marc-145 cells were transfected with either a vector or Myc-NUDT7, then infected with PRRSV (MOI=1) for 12, 24, and 36 hours. PRRSV-N mRNA and protein levels were quantified using RT-qPCR and western blot analysis. Normalization was performed using β-actin as the internal control. (C) Marc-145 cells underwent identical treatment conditions, and the supernatant was collected for TCID 50 analysis to assess viral titer. (D) Marc-145 cells were transfected with either siNC or siNUDT7 and subsequently infected with PRRSV at a multiplicity of infection (MOI) of 1. Western blot analysis was used to assess PEDV-N protein levels. (E) The N protein was visualized using an anti-N antibody (green), while the nuclei were counterstained blue. Fluorescence images were taken at 10x magnification (F-G) Sequence analysis was conducted on both wild-type (WT) and CRISPR-Cas9-mediated NUDT7 knockout (KO) iPAM cell lines. Additionally, the cell viability and growth kinetics of NUDT7 KO cells were compared with those of WT cells. (H-I) Wild-type (WT) and NUDT7 knockout (KO) cells were infected with PRRSV, followed by analysis of cell lysates using RT-qPCR and immunoblotting. (J) PRRSV-N levels were assessed via immunofluorescence in NUDT7 KO cells 24 hours post-PRRSV infection. (K) The schematic diagram of the truncated domain of NUDT7. (L) Wild-type (WT) or NUDT7 knockout (KO) cells were transfected with either full-length or truncated NUDT7 for 24 hours, followed by infection with PRRSV at a multiplicity of infection (MOI) of 1 for another 24 hours. Subsequent cell lysates were subjected to immunoblot analysis.

Article Snippet: HEK293T (ATCC, CRL-11268) and Marc-145 (ATCC, CRL-12231) cells were maintained in DMEM (Gibco, C11995500BT) with 10% FBS (PAN-Biotech, P30-1406). iPAMs, an immortalized porcine alveolar macrophage cell line expressing the PRRSV receptor CD163, were cultured in RPMI 1640 medium (Gibco, catalog no.).C22400500BT) with 10% fetal bovine serum (FBS) added.

Techniques: Infection, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control, Fluorescence, Sequencing, CRISPR, Knock-Out, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: NUDT7 Modulates the UBA52-SREBF1 Signaling Axis to Promote PRRSV Replication via Lipid Synthesis

doi: 10.7150/ijbs.127844

Figure Lengend Snippet: NUDT7 reprograms PRRSV-induced lipid metabolism. (A-B) Marc-145 cells, along with empty vector and Myc-NUDT7 transfected cells, were exposed to PRRSV for 24 hours. WT and NUDT7 KO cells were as described above (B). Oil Red O staining was used to analyze LDs. Scale bar: 10 μm. (C-D) LDs were detected by BODIPY staining. (E) RT-qPCR analysis of NUDT7-overexpressing Marc-145 cells revealed mRNA expression levels of several lipid metabolism-related factors. (F-I) PRRSV infected OE-NUDT7 Marc-145 cells or NUDT7-KO iPAM cells respectively. Cellular lipids were extracted, and TG and TC levels were quantified by biochemical kits. (J-K) PRRSV infected OE-NUDT7 Marc-145 cells or NUDT7-KO iPAM cells at specified time points, respectively. Analysis of lipid metabolism-related factors using Western blot.

Article Snippet: HEK293T (ATCC, CRL-11268) and Marc-145 (ATCC, CRL-12231) cells were maintained in DMEM (Gibco, C11995500BT) with 10% FBS (PAN-Biotech, P30-1406). iPAMs, an immortalized porcine alveolar macrophage cell line expressing the PRRSV receptor CD163, were cultured in RPMI 1640 medium (Gibco, catalog no.).C22400500BT) with 10% fetal bovine serum (FBS) added.

Techniques: Plasmid Preparation, Transfection, Staining, Quantitative RT-PCR, Expressing, Infection, Western Blot

Journal: International Journal of Biological Sciences

Article Title: NUDT7 Modulates the UBA52-SREBF1 Signaling Axis to Promote PRRSV Replication via Lipid Synthesis

doi: 10.7150/ijbs.127844

Figure Lengend Snippet: SREBF1 emerges as a critical regulator in NUDT7-dependent lipogenesis. (A) NUDT7 KO iPAM cells were treated with lipid (cholesterol and oleic acid) dose-dependent therapy and infected with PRRSV (MOI=1) for 24 h. The cell lysates were analyzed by immunoblotting. (B) The schematic diagram illustrates the experiment in which siSREBF1 cells were complemented with empty or NUDT7 and infected with PRRSV and the knockdown efficiency of SREBF1. (C) The triglyceride (TG) levels in siSREBF1 Marc-145 cells, transfected with either an empty vector or NUDT7 and subsequently infected with PRRSV, were analyzed. (D-E) Marc-145 was transfected with siRNA or siSREBF1 and complemented with NUDT7. Lipid droplets were stained with oil red O and Biodipy 24 hours post-PRRSV infection. Scale bar: 10 μm. (F-G) As previously described, PRRSV infection occurred after overexpression of SREBF1 in NUDT7 KO iPAM cells for 24 h. Cell lipid droplets are indicated with Oil Red O and Biodipy. Scale bar: 10 μm. (H-J) Marc-145 cells were transfected with siSREBF1 and subsequently infected with PRRSV at a designated time. PRRSV-N expression was analyzed using RT-qPCR, western blot, and immunofluorescence. (K) iPAM cells were exposed to PRRSV at an MOI of 1 and subsequently treated with either 10 µM Fatostatin or a DMSO vehicle control at a 1:1,000 dilution.

Article Snippet: HEK293T (ATCC, CRL-11268) and Marc-145 (ATCC, CRL-12231) cells were maintained in DMEM (Gibco, C11995500BT) with 10% FBS (PAN-Biotech, P30-1406). iPAMs, an immortalized porcine alveolar macrophage cell line expressing the PRRSV receptor CD163, were cultured in RPMI 1640 medium (Gibco, catalog no.).C22400500BT) with 10% fetal bovine serum (FBS) added.

Techniques: Infection, Western Blot, Knockdown, Transfection, Plasmid Preparation, Staining, Over Expression, Expressing, Quantitative RT-PCR, Immunofluorescence, Control

Journal: International Journal of Biological Sciences

Article Title: NUDT7 Modulates the UBA52-SREBF1 Signaling Axis to Promote PRRSV Replication via Lipid Synthesis

doi: 10.7150/ijbs.127844

Figure Lengend Snippet: NUDT7 targets UBA52 to enhance proteasomal degradation. (A) Marc-145 cells were either transfected with an empty vector or FLAG-NUDT7 for 36 hours, followed by PRRSV infection (MOI=1) for 24 hours. Subsequently, the cells were lysed, and the supernatant from the lysate was analyzed using anti-Flag magnetic beads, with identification carried out through mass spectrometry. The proteins with high peaks that potentially interact with NUDT7 were enumerated. (B) GO enrichment analysis diagram. (C) KEGG pathway enrichment analysis results. (D) Interaction network graph illustrating protein-protein interactions (PPI). (E) Co-immunoprecipitation (Co-IP) assay was conducted in HEK293T cells, which were transfected with Myc-UBA52 and either Flag-NUDT7 or a control vector. After the transfection, cell lysates underwent immunoprecipitation using a Flag antibody followed by immunoblotting with Myc and Flag antibodies. (F) Molecular docking simulation of NUDT7 and UBA52 was performed utilizing ZDOCK software. (G) Co-immunoprecipitation (Co-IP) using anti-UBA52 and immunoblotting analysis were conducted on protein lysates from both control and iPAM infected with PRRSV (MOI of 5, 24 hours). (H) Infected Marc-145 cells with PRRSV (MOI of 1, 24 h) and control were labeled with the specified antibodies and examined through confocal microscopy. The UBA52 signal is shown in red, while NUDT7 is in green. The scale bars measure 20 μm. (I) Marc-145 cells were transfected with Myc-tagged NUDT7 for 24 hours and then subjected to PRRSV infection for 0, 12, and 24 hours. (J) An immunoblot was performed on protein extracts derived from Marc-145 cells that had been transfected with plasmids for Myc-UBA52 along with various amounts of Flag-NUDT7. (K) The UBA52 mRNA levels in Marc-145 cells transfected with Myc-UBA52 and differing quantities of Flag-NUDT7 were assessed through RT-qPCR analysis. Results are shown as relative fold changes, normalized against β-actin controls. (L) An analysis of protein extracts was carried out on Marc-145 cells transfected with plasmids encoding Myc-NUDT7 and its truncated forms. (M) The specified plasmid was transfected into Marc-145 for 24 hours. After being infected with PRRSV for an additional 24 hours, lipid droplets were visualized using Bodipy staining. Scale bar: 10 μm. (N-P) Immunoblot analysis on lysates from Marc-145 co-transfected for 24 hours with Flag-NUDT7 and Myc-UBA52 was performed, followed by treatment with various inhibitors 24 hours before PRRSV infection: MG-132 (10µM) (N), chloroquine (CQ, 20µM) (P), or NH4Cl (20 mM) (Q).

Article Snippet: HEK293T (ATCC, CRL-11268) and Marc-145 (ATCC, CRL-12231) cells were maintained in DMEM (Gibco, C11995500BT) with 10% FBS (PAN-Biotech, P30-1406). iPAMs, an immortalized porcine alveolar macrophage cell line expressing the PRRSV receptor CD163, were cultured in RPMI 1640 medium (Gibco, catalog no.).C22400500BT) with 10% fetal bovine serum (FBS) added.

Techniques: Transfection, Plasmid Preparation, Infection, Magnetic Beads, Mass Spectrometry, Protein-Protein interactions, Co-Immunoprecipitation Assay, Control, Immunoprecipitation, Western Blot, Software, Labeling, Confocal Microscopy, Derivative Assay, Quantitative RT-PCR, Staining

Journal: International Journal of Biological Sciences

Article Title: NUDT7 Modulates the UBA52-SREBF1 Signaling Axis to Promote PRRSV Replication via Lipid Synthesis

doi: 10.7150/ijbs.127844

Figure Lengend Snippet: NUDT7 enhances the stability of SREBF1 by inhibiting UBA52-induced polyubiquitination and subsequent degradation of SREBF1. (A) Co-immunoprecipitation (Co-IP) assays were conducted in Marc-145 cells that were transfected with Myc-UBA52 and Flag-SREBF1 or control vectors. After 24 hours of transfection, cells were inoculated with PRRSV (MOI=5), and cell lysates were subjected to immunoprecipitation using Flag antibodies, followed by immunoblotting with Myc and Flag antibodies. (B) Examination of UBA52 and SREBF1 interactions through Co-IP in Marc-145 cells. (C) The distribution of SREBF1 and UBA52 in cells infected with PRRSV was assessed via immunofluorescence. Scale bar: 20 μm. (D) The levels of specific proteins in Marc-145 cells, either with UBA52 overexpression or subjected to PRRSV infection, were analyzed using Western blotting. (E) RT-qPCR was performed to evaluate the mRNA levels of SREBF1 in Marc-145 cells transfected with plasmids expressing Myc-UBA52. Results are shown as relative fold changes, normalized against β-actin controls. (F-G) Cells were treated with CHX (15 μM) for the specified duration, and the expression of SREBF1 was evaluated in the relevant groups. (H-I) The ubiquitination of SREBF1 was identified through Western blot analysis in both UBA52-overexpressing Marc-145 cells and UBA52 knockdown iPAM cells infected with PRRSV. (J-L) The examination of SREBF1 ubiquitination was conducted on Marc-145 cells that were transfected with specific constructs and subsequently infected with PRRSV. (M) Endogenous SREBF1 expression was detected via Western blotting after inoculating PRRSV 24 hours following the overexpression of the specified plasmids. (N) BODIPY staining showed the presence of lipid droplets in cells 24 hours post-PRRSV infection following the overexpression of the indicated plasmid in Marc-145 cells (left). Scale bar: 10 μm. The relative fluorescence intensity of BODIPY in the cells was quantified (right). (O) For 24 hours, Marc-145 cells were transfected with a Flag-tagged NUDT7 plasmid. Following cell lysis and centrifugation, the supernatant was incubated overnight with SREBF1 magnetic beads, and subsequent protein immunoblots were performed to detect proteins.

Article Snippet: HEK293T (ATCC, CRL-11268) and Marc-145 (ATCC, CRL-12231) cells were maintained in DMEM (Gibco, C11995500BT) with 10% FBS (PAN-Biotech, P30-1406). iPAMs, an immortalized porcine alveolar macrophage cell line expressing the PRRSV receptor CD163, were cultured in RPMI 1640 medium (Gibco, catalog no.).C22400500BT) with 10% fetal bovine serum (FBS) added.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Control, Western Blot, Infection, Immunofluorescence, Over Expression, Quantitative RT-PCR, Expressing, Ubiquitin Proteomics, Knockdown, Construct, Staining, Plasmid Preparation, Fluorescence, Lysis, Centrifugation, Incubation, Magnetic Beads

Journal: International Journal of Biological Sciences

Article Title: NUDT7 Modulates the UBA52-SREBF1 Signaling Axis to Promote PRRSV Replication via Lipid Synthesis

doi: 10.7150/ijbs.127844

Figure Lengend Snippet: NUDT7 diminishes the IFN-I immune response during viral infection. (A-C) Luciferase reporter assays were conducted to evaluate the promoter activity of IFN-β, ISRE, and NF-kB in HEK293T cells that were transfected with escalating amounts of Myc-NUDT7 or an empty vector (EV) for 24 hours, subsequently treated with or without VSV for 18 hours. (D-F) Immunoblot analysis (D) of both total and phosphorylated IRF3, along with RT-qPCR assessments (E-F) of specified gene expression in HEK293T cells that underwent transfection with Myc-NUDT7 or EV for 24 hours, followed by VSV infection at defined time points. (G-H) Analysis using fluorescence microscopy (G) and flow cytometry (H) to investigate the replication of GFP-VSV in HEK293T cells transfected with either EV or varying amounts of Myc-NUDT7 at specified dosages for 24 hours, followed by treatment with or without GFP-VSV infection at designated time intervals. (I-L) Marc-145 cells were transfected with either EV or Myc-NUDT7 for 24 hours and subsequently infected with PRRSV. Cells were collected at 12-, 24-, and 36-hours post-infection (hpi) for RT-qPCR to assess mRNA expression levels (I-K) or for western blotting to determine protein expression levels (L).

Article Snippet: HEK293T (ATCC, CRL-11268) and Marc-145 (ATCC, CRL-12231) cells were maintained in DMEM (Gibco, C11995500BT) with 10% FBS (PAN-Biotech, P30-1406). iPAMs, an immortalized porcine alveolar macrophage cell line expressing the PRRSV receptor CD163, were cultured in RPMI 1640 medium (Gibco, catalog no.).C22400500BT) with 10% fetal bovine serum (FBS) added.

Techniques: Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Gene Expression, Fluorescence, Microscopy, Flow Cytometry, Expressing

Journal: Journal of Pharmacokinetics and Pharmacodynamics

Article Title: Population pharmacokinetics and exposure–response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia

doi: 10.1007/s10928-018-9592-y

Figure Lengend Snippet: Summary of the studies and data included in the analyses

Article Snippet: Microplate wells coated with a mouse anti-evolocumab monoclonal antibody (clone no. 1.18.1, Amgen Inc.) were used to capture evolocumab from serum.

Techniques: Sampling

Journal: Journal of Pharmacokinetics and Pharmacodynamics

Article Title: Population pharmacokinetics and exposure–response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia

doi: 10.1007/s10928-018-9592-y

Figure Lengend Snippet: Evolocumab pharmacokinetic and exposure–response model. a Pharmacokinetic model; k a absorption rate constant; k el elimination rate constant; k m concentration of half-maximal nonlinear clearance; V max nonlinear clearance capacity. b Exposure–response model; Eff LDL-C lowering effect; E max theoretical maximum evolocumab response for the average of weeks 10 and 12; EC 50 ((µg/mL) * day) AUC wk8–12 (Q2W) to achieve half-maximal response; REG regimen effect on EC 50 with an indicator variable, i , with values of 0 or 1 was used indicate Q2W or QM regimens

Article Snippet: Microplate wells coated with a mouse anti-evolocumab monoclonal antibody (clone no. 1.18.1, Amgen Inc.) were used to capture evolocumab from serum.

Techniques: Concentration Assay

Journal: Journal of Pharmacokinetics and Pharmacodynamics

Article Title: Population pharmacokinetics and exposure–response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia

doi: 10.1007/s10928-018-9592-y

Figure Lengend Snippet: Time-course of model-predicted and observed serum evolocumab concentrations after doses of 140 mg SC Q2W (6 doses) and 420 mg SC QM (3 doses). Simulations were performed (number of trials = 100) on the entire dataset. Dots: observed evolocumab serum concentrations. Panels a, b, and c: blue shaded area: 90% prediction interval of simulated evolocumab serum concentration–time profile, and red line is predicted median, whereas black line is observed median. Panel d: The solid red lines represent the 95th (upper red line), 50th (middle red line), and 5th (lower red line) percentiles of the observed prediction-corrected serum concentration. The observed prediction-corrected plasma concentrations are represented by grey circles. The black lines (upper: 95th, middle: 50th, and lower: 5th) represent the simulation-based prediction, and the surrounding semitransparent blue field represents a simulation-based 90% prediction interval for the corresponding simulation-based prediction intervals. Q2W once every 2 weeks; QM once monthly; SC subcutaneous

Article Snippet: Microplate wells coated with a mouse anti-evolocumab monoclonal antibody (clone no. 1.18.1, Amgen Inc.) were used to capture evolocumab from serum.

Techniques: Concentration Assay

Journal: Journal of Pharmacokinetics and Pharmacodynamics

Article Title: Population pharmacokinetics and exposure–response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia

doi: 10.1007/s10928-018-9592-y

Figure Lengend Snippet: Observed and model-predicted serum evolocumab concentrations. Solid line: line of unity; dashed line: locally weighted scatterplot smoothing (LOWESS); PRED population predicted concentration; IPRED individual predicted concentration

Article Snippet: Microplate wells coated with a mouse anti-evolocumab monoclonal antibody (clone no. 1.18.1, Amgen Inc.) were used to capture evolocumab from serum.

Techniques: Concentration Assay

Journal: Journal of Pharmacokinetics and Pharmacodynamics

Article Title: Population pharmacokinetics and exposure–response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia

doi: 10.1007/s10928-018-9592-y

Figure Lengend Snippet: Parameter estimates for exposure–response model

Article Snippet: Microplate wells coated with a mouse anti-evolocumab monoclonal antibody (clone no. 1.18.1, Amgen Inc.) were used to capture evolocumab from serum.

Techniques:

Journal: Journal of Pharmacokinetics and Pharmacodynamics

Article Title: Population pharmacokinetics and exposure–response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia

doi: 10.1007/s10928-018-9592-y

Figure Lengend Snippet: Observed data and 90% prediction interval for week 10 and 12 mean calculated LDL-C for phase 2 studies by weeks 8–12 evolocumab-predicted AUC. Prediction of the mean week 10 and 12 calculated LDL-C concentration, in percentage change from baseline, 50th (solid line) and 5th and 95th (dashed lines) percentiles. Simulations were performed for n = 2000 patients. Points: observed individual mean of weeks 10 and 12 LDL-C measurements. Vertical line: mean observed AUC wk8–12 in phase 2. %CFB percentage change from baseline; AMG 145 evolocumab; AUC area under the concentration–time curve; LDL - C low-density lipoprotein-cholesterol; Q2W once every 2 weeks; QM once monthly

Article Snippet: Microplate wells coated with a mouse anti-evolocumab monoclonal antibody (clone no. 1.18.1, Amgen Inc.) were used to capture evolocumab from serum.

Techniques: Concentration Assay

Journal: Journal of Pharmacokinetics and Pharmacodynamics

Article Title: Population pharmacokinetics and exposure–response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia

doi: 10.1007/s10928-018-9592-y

Figure Lengend Snippet: Forest plots of covariate effects with 95% CI for evolocumab AUC wk8–12 for 140 mg SC Q2W and 420 mg SC QM. The statin covariate represents patients only taking a statin and no other concomitant medication. The statin + Ezet covariate includes all patients on Ezet, regardless of concomitant medications. For patients in the pharmacokinetics model, 93% of those taking Ezet were also taking a statin; thus, the Ezet covariate most generally represents a combination (statin + Ezet) therapy covariate. AUC area under the time-concentration curve; CI confidence interval; Ezet ezetimibe; PCSK9 BL proprotein convertase subtilisin/kexin type 9 baseline (low, 4.8 nM [355 ng/mL]; high, 8.1 nM [599 ng/mL]); Q2W once every 2 weeks; QM once monthly; SC subcutaneous

Article Snippet: Microplate wells coated with a mouse anti-evolocumab monoclonal antibody (clone no. 1.18.1, Amgen Inc.) were used to capture evolocumab from serum.

Techniques: Concentration Assay

Journal: Journal of Pharmacokinetics and Pharmacodynamics

Article Title: Population pharmacokinetics and exposure–response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia

doi: 10.1007/s10928-018-9592-y

Figure Lengend Snippet: Forest plots of covariate effects with 95% CI for evolocumab week 10 and 12 mean calculated LDL-C lowering for 140 mg SC Q2W and 420 mg SC QM. CI confidence interval; Ezet ezetimibe; HeFH heterozygous familial hypercholesterolemia; LDL - C low-density lipoprotein-cholesterol; PCSK9 BL proprotein convertase subtilisin/kexin type 9 baseline (low, 4.8 nM [355 ng/mL]; high, 8.1 nM [599 ng/mL]); Q2W once every 2 weeks; QM once monthly; SC subcutaneous

Article Snippet: Microplate wells coated with a mouse anti-evolocumab monoclonal antibody (clone no. 1.18.1, Amgen Inc.) were used to capture evolocumab from serum.

Techniques:

Journal: Nature Communications

Article Title: Construction of a synthetic methodology-based library and its application in identifying a GIT/PIX protein–protein interaction inhibitor

doi: 10.1038/s41467-022-34598-7

Figure Lengend Snippet: a Kinetic measurement of the binding affinity of 14-5-18 to GIT1 by BLI assay. Concentrations ranging from 25 to 400 μM were shown with the real-time response for each step of the kinetic assay. b Co-IP assay to indicate the interaction of GIT1 and β-Pix and the inhibitory effects of 14-5-18 . HA-GIT1 and Flag-β-Pix were transfected into HEK-293T cells, and the cell lysates were collected to perform the co-IP assay at concentrations of 0, 5, 20, and 50 μM. The demonstrated blots were representatives from three independent experiments with similar results. c Fluorescence colocalization experiment to visualize the cell distribution of GIT1 and β-Pix with or without treatment of 14-5-18 . GFP-GIT1 (green) and Myc-β-Pix were expressed followed by treatment with a fluorescent secondary antibody (red) in the MGC803 cell line to construct the fluorescence system. The cells were observed under confocal microscopy after 14-5-18 was added at concentrations of 0 and 20 μM. Plot fluorescence intensities of the area marked by the white lines in the left panels are shown in the right graphs. Green and red curves represent the GIT1 and β-Pix, respectively. Scale bar = 10 μm. The demonstrated figures were representatives from three independent experiments with similar results. d The percentage of GIT1/β-Pix colocalized cells with or without treatment of 14-5-18 . The analysis was performed in n = 30 cells from three independent experiments. Statistical analysis: Two-sided Fisher’s exact test ( d ). Source data are provided as a Source Data file.

Article Snippet: The direct interaction between 14-5-18 and GIT1 was detected using ForteBio Octet RED (Sartorius, Germany).

Techniques: Binding Assay, Kinetic Assay, Co-Immunoprecipitation Assay, Transfection, Fluorescence, Construct, Confocal Microscopy