Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Hemoglobin production in BL21(DE3) transformed with various plasmids. (A) Absorbance scans of live cultures of BL21(DE3) with and without hemoglobin or P. shigelloides heme transport genes. Solid line, E. coli containing hemoglobin and heme transport genes [(BL21(DE3)/pHB0.0, pHUG21]; dotted line, E. coli containing hemoglobin genes alone [BL21(DE3)/pHB0.0, pWSK29]; dashed line, E. coli containing heme transport genes alone [BL21(DE3)/pDSV1, pHUG21]. Values on the x axis are wavelengths in nanometers. (B) First derivative spectrum obtained from absorbance scans. Derivative values are shown beside each arrow. Solid line, BL21(DE3) containing heme transport and hemoglobin genes; dotted line, BL21(DE3) containing hemoglobin genes alone. (C) Immunoblot of soluble (S) and insoluble (I) fractions from 0.004 OD unit of cells at a wavelength of 700 nm. Lanes 1 and 4, BL21(DE3) containing heme transport genes alone; lanes 2 and 5, BL21(DE3) containing hemoglobin genes alone; lanes 3 and 6, BL21(DE3) containing hemoglobin and heme transport genes; lane 7, 0.45 μg of hemoglobin (Sigma Chemicals).

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Transformation Assay, Western Blot

Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Hemoglobin production in BL21(DE3) containing the one-plasmid or two-plasmid system. (A) Genetic and partial restriction enzyme map of genes on pHB0.0hug. Genes are indicated with horizontal arrows, and promoters are indicated with vertical bent arrows. Restriction enzyme sites are as follows: B, BamHI; H, HindIII; N, NheI; S, SalI; and W, BsiWI. (B) Immunoblot of soluble (S) and insoluble (I) fractions. Lanes 1 and 3, BL21(DE3) containing the two-plasmid system (pHB0.0, pHUG21); lanes 2 and 4, BL21(DE3) containing the one-plasmid system (pHB0.0hug); lane 5, 0.45 μg of hemoglobin. (C) Densitometry analysis of immunoblots of soluble fractions. Data are from three independent sets of samples analyzed on the same immunoblot; soluble fractions from 0.004 OD unit of cells at a wavelength of 700 nm were loaded. Open circles, hemoglobin control; filled squares, BL21(DE3) containing the two-plasmid system; filled triangles, BL21(DE3) containing the one-plasmid system.

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Plasmid Preparation, Western Blot, Control

Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Analysis of hemoglobin production in BL21(DE3) containing various plasmids

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Plasmid Preparation

Journal: Frontiers in Genetics

Article Title: SYK Is Associated With Malignant Phenotype and Immune Checkpoints in Diffuse Glioma

doi: 10.3389/fgene.2022.899883

Figure Lengend Snippet: Expression pattern of SYK by single-cell sequencing analysis and the association between SYK expression and M2 macrophage infiltration. (A,B) SYK expression in the different cell types; (B–F) The correlation between SYK expression and CD163 and VSIG4 expression in the four cohorts. (G,H) The correlation between SYK expression and CD163 expression by IHC staining.

Article Snippet: Immunohistochemistry (IHC) was performed using a primary antibody against PD-L1 (Cell Signaling Technology Pathways, United States), CD163 (Proteintech, China), and SYK (Proteintech, China).

Techniques: Expressing, Sequencing, Immunohistochemistry

Journal: iScience

Article Title: A cyclic peptide-grafted Fc with hepatocyte growth factor functionality ameliorates hepatic fibrosis in a non-alcoholic steatohepatitis mouse model

doi: 10.1016/j.isci.2024.110426

Figure Lengend Snippet:

Article Snippet: The serum and lysate buffer concentrations of Fc and Fc(mML1)B3 were determined using a human immunoglobulin recognition immunoassay (human IgG ELISA quantitation set, Bethyl Laboratories, Montgomery, USA).

Techniques: Recombinant, Expressing, Endotoxin Assay, Acid Assay, Lysis, Phospho-proteomics, Ab Array, MTS Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Reverse Transcription, Staining, TUNEL Assay, Activity Assay, RNA Sequencing, Gene Expression, Software, Immunohistochemistry, Flow Cytometry, Imaging, Microscopy, Next-Generation Sequencing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Journal of biomolecular screening

Article Title: Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

doi: 10.1177/1087057113499776

Figure Lengend Snippet: Figure 1. Method for obtaining day 7 erythroid progenitor cells (EPCs) for use in γ-globin screening assay. EPCs were generated and expanded from cryopreserved human bone marrow CD34+ cells in a two-phase liquid culture system. Expansion media with low erythropoietin (EPO) (0.5 U/mL) were used from days 1 to 7 of culture. Day 7 EPCs were then cultured in media with high EPO (3 U/mL) for an additional 3 days in the presence or absence of tool or test compounds to assess γ-globin–inducing potential by various methods (i.e., flow cytometry, enzyme-linked immunosorbent assay [ELISA]). HCI, high-content imaging; IL-2, interleukin 3; SCF, stem cell factor.

Article Snippet: Hoechst 33342 (cat. H3570) and FITC-conjugated mouse anti–human HbF monoclonal Ab for flow cytometry (anti–HbF-FITC; cat. MHFH01) were purchased from Life Technologies (Carlsbad, CA). γ-Globin calibrator (cat. RC80-135) and anti-HbF sheep polyclonal Ab for enzyme-linked immunosorbent assay (ELISA) (cat. A80-136A) were purchased from Bethyl Lab (Montgomery, TX), and Dulbecco’s phosphate-buffered saline (DPBS) buffer was from Mediatech (Manassas, VA; cat. 21-031-LB).

Techniques: Screening Assay, Generated, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Imaging

Journal: Journal of biomolecular screening

Article Title: Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

doi: 10.1177/1087057113499776

Figure Lengend Snippet: Figure 3. Development and optimization of 384-well enzyme-linked immunosorbent assay (ELISA). (A). γ-Globin calibrator standard curves in the ELISA. ELISA plates were coated with sheep anti–γ-globin Ab and blocked with blocking buffer, then washed prior to the addition of cell lysates. Plates were coated and blocked on the same day of lysate binding (); plates were coated, blocked, washed, and stored at 4 °C for 3 days before lysate binding (); plates were coated, blocked, washed, and stored at 4 °C for 20 days before lysate binding (∆). (B) Cell density titration using decitabine as a tool compound. Day 7 erythroid progenitor cell (EPCs) were treated under different cell densities for 72 h. γ-Globin protein in treated cell lysates was determined by ELISA. The percent induction of decitabine was normalized against DMSO control (100%). 2000 cells/well (); 3000 cells/well (); 4000 cells/well (); 5000 cells/well (∆). (C) γ-Globin response of cells treated with compounds. Day 7 EPCs were treated with the tool compounds at different doses for 72 h. Treated cells were then lysed and cell lysates were subjected to γ-globin protein detection by ELISA. The percent induction was normalized against control (100%). Decitabine (); hydroxyurea (). Error bars are ± standard deviations of duplicate samples.

Article Snippet: Hoechst 33342 (cat. H3570) and FITC-conjugated mouse anti–human HbF monoclonal Ab for flow cytometry (anti–HbF-FITC; cat. MHFH01) were purchased from Life Technologies (Carlsbad, CA). γ-Globin calibrator (cat. RC80-135) and anti-HbF sheep polyclonal Ab for enzyme-linked immunosorbent assay (ELISA) (cat. A80-136A) were purchased from Bethyl Lab (Montgomery, TX), and Dulbecco’s phosphate-buffered saline (DPBS) buffer was from Mediatech (Manassas, VA; cat. 21-031-LB).

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Titration, Control

Journal: Journal of biomolecular screening

Article Title: Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

doi: 10.1177/1087057113499776

Figure Lengend Snippet: Figure 6. γ-Globin induction response and effects on cell viability by compound A in day 7 erythroid progenitor cell (EPCs). The γ-globin induction abilities of compound A were monitored in a dose-dependent manner in the enzyme-linked immunosorbent assay (ELISA). Cell viability was measured using the CellTiter-Glo (Promega, Madison, WI) assay. Each data point is an average of nine or four testings on separate dates at an indicated concentration in ELISA or CellTiter-Glo assays, respectively. Fetal hemoglobin ELISA (); cell viability (). Error bars are ± standard deviations.

Article Snippet: Hoechst 33342 (cat. H3570) and FITC-conjugated mouse anti–human HbF monoclonal Ab for flow cytometry (anti–HbF-FITC; cat. MHFH01) were purchased from Life Technologies (Carlsbad, CA). γ-Globin calibrator (cat. RC80-135) and anti-HbF sheep polyclonal Ab for enzyme-linked immunosorbent assay (ELISA) (cat. A80-136A) were purchased from Bethyl Lab (Montgomery, TX), and Dulbecco’s phosphate-buffered saline (DPBS) buffer was from Mediatech (Manassas, VA; cat. 21-031-LB).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Nature Communications

Article Title: Long-term statins administration exacerbates diabetic nephropathy via ectopic fat deposition in diabetic mice

doi: 10.1038/s41467-023-35944-z

Figure Lengend Snippet: All mice were ~50 weeks old. a Schematic diagram showing the procedure of long-term administration of statins for db/db mice. b Body weight. For 12 weeks, n = 6 biologically independent mice in Db/m group, n = 13 in Db group, n = 10 in Db + Ato5 group, n = 16 in Db + Ato10 group, n = 9 in Db + Rosu20 group. But over time, some of the mice gradually lost weight and died. By 43W, n = 6 biologically independent mice in Db/m group, n = 6 in Db group, n = 5 in Db + Ato5 group, n = 3 in Db + Ato10 group, n = 3 in Db + Rosu20 group. For specific values see as a “Source Data file”. c Kaplan–Meier survival curves of db/db mice after long-term administration of statin. Log-rank (Mantel–Cox) test was used for the analysis of statistical significance. Exact p value = 4 × 10 −7 . d Measurements of fasting blood glucose. n = 4 per time point. Significance ** p < 0.01 compared with Db + Ato5 group, ### p < 0.001 compared with Db + Ato10 group; e GHbA1c levels. n = 6 in each group. f ITT and AUC for db / db mice at 40 weeks. n = 4 per time point, and n = 5 per time point i n Db + Ato5 group. g Immunohistochemical images of the RAGE in kidney sections. RAGE positive staining was mainly localized in the plasma membrane domains, and arrows represent RAGE expression in the cytoplasm of renal tubular epithelial cells . Original magnification ×400. Scale bar: 50 µm. All image part of the kidney was cortex. Renal structures indicated as proximal tubule (PT). h Quantitative analysis of expression of RAGE. n = 6 in each group. Data are expressed as means ± SEM ( b , d , e , f , h ). Significance tests were two-tailed, one-way ANOVA followed by Tukey’s test was performed ( b , d – f , h ). Source data are provided as a Source Data file.

Article Snippet: The serum fasting insulin levels and Glycated Hemoglobin A1c (GHbA1c) levels were examined using an ELISA kit (E08141m, Cusabio, Wuhan, China).

Techniques: Immunohistochemical staining, Staining, Clinical Proteomics, Membrane, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: Label-free quantitation of glycated hemoglobin in single red blood cells by transient absorption microscopy and phasor analysis

doi: 10.1126/sciadv.aav0561

Figure Lengend Snippet: ( A and B ) Protein structure of the glycation site of HbA1c, and normal Hb, respectively. The crystal structure of HbA1c (3B75) and Hb (1LFZ) are from Protein Data Bank. Cyan sphere, water molecule; red sphere, oxygen atom; green sphere, carbon atom; blue sphere, nitrogen atom. In (A), the polar force between glucose and heme and surrounding water is highlighted by blue dashed lines. Regions of interest (ROIs) are highlighted by red dashed circles. ( C and D ) Zoom-in view of the interaction between glucose and porphyrin ring from glycated Hb (C) and normal Hb (D).

Article Snippet: Transient absorption imaging of whole blood study was achieved by measuring the HbA1c fraction from type 2 diabetic human whole blood (Lee Biosolutions, Inc.) and healthy human whole blood (Boston Children’s Hospital Blood Donor Center).

Techniques:

Journal: Science Advances

Article Title: Label-free quantitation of glycated hemoglobin in single red blood cells by transient absorption microscopy and phasor analysis

doi: 10.1126/sciadv.aav0561

Figure Lengend Snippet: ( A and B ) Fluorescence spectra of Hb (0.025 mg/ml) (A) along with HbA1c (0.025 mg/ml) (B), respectively. Excitation wavelength, 447 nm; integration time, 1000 s; band-pass filter, 488 ± 10 nm; power on the sample, 150 μW. 20× air objective. ( C and D ) Time-resolved photoluminescence (PL) measurements of Hb (0.025 mg/ml) (C) and HbA1c (0.025 mg/ml) (D), respectively. a.u., arbitrary units. ( E and F ) Absorption spectra (normalized) of Hb (E) and HbA1c (F), respectively.

Article Snippet: Transient absorption imaging of whole blood study was achieved by measuring the HbA1c fraction from type 2 diabetic human whole blood (Lee Biosolutions, Inc.) and healthy human whole blood (Boston Children’s Hospital Blood Donor Center).

Techniques: Fluorescence

Journal: Science Advances

Article Title: Label-free quantitation of glycated hemoglobin in single red blood cells by transient absorption microscopy and phasor analysis

doi: 10.1126/sciadv.aav0561

Figure Lengend Snippet: ( A and B ) Time-resolved decay curves (normalized) of Hb (A) and HbA1c (B), respectively. int., intensity. ( C ) Merged time-resolved curves (normalized) of Hb and HbA1c. ( D and E ) Time-resolved decay curves (normalized) of oxyHb (D) and oxyHbA1c (E), respectively. ( F ) Merged time-resolved curves (normalized) of oxyHb and oxyHbA1c. ( G ) Proposed excited-state dynamic pathway of Hb when pumped at 520 nm and probed at 780 nm.

Article Snippet: Transient absorption imaging of whole blood study was achieved by measuring the HbA1c fraction from type 2 diabetic human whole blood (Lee Biosolutions, Inc.) and healthy human whole blood (Boston Children’s Hospital Blood Donor Center).

Techniques:

Journal: Science Advances

Article Title: Label-free quantitation of glycated hemoglobin in single red blood cells by transient absorption microscopy and phasor analysis

doi: 10.1126/sciadv.aav0561

Figure Lengend Snippet: ( A ) Time-resolved decay curves (normalized) of standard HbA1c solutions (human whole blood based) at different concentrations. ( B ) Zoom-in view of (A) from delay time of 1 to 4 ps. ( C ) Component s versus different ω from 0 to 2π THz for pure HbA1c and Hb. ( D ) Phasor plot of standard HbA1c solutions of different concentrations when ω = 0.8π THz. ( E ) Calibration curve of standard HbA1c solution at different concentrations (component s versus HbA1c%).

Article Snippet: Transient absorption imaging of whole blood study was achieved by measuring the HbA1c fraction from type 2 diabetic human whole blood (Lee Biosolutions, Inc.) and healthy human whole blood (Boston Children’s Hospital Blood Donor Center).

Techniques:

Journal: Science Advances

Article Title: Label-free quantitation of glycated hemoglobin in single red blood cells by transient absorption microscopy and phasor analysis

doi: 10.1126/sciadv.aav0561

Figure Lengend Snippet: ( A ) Pseudocolor transient absorption images (delay time, 0 ps) of single RBCs with ROIs are highlighted by blue dashed circles. Scale bar, 10 μm. Pump: 520 nm, 2 mW on the sample; probe: 780 nm, 10 mW on the sample. int., intensity. ( B and C ) Time-resolved decay curves (normalized) of ROIs shown in (A). ( D to F ) HbA1c fraction (in single RBCs) distribution along with the fitted glucose concentration from three diabetic whole blood samples. ( G to I ) HbA1c fraction (in single RBCs) distribution along with the derived glucose concentration from three healthy whole blood samples. Curve fitted by .

Article Snippet: Transient absorption imaging of whole blood study was achieved by measuring the HbA1c fraction from type 2 diabetic human whole blood (Lee Biosolutions, Inc.) and healthy human whole blood (Boston Children’s Hospital Blood Donor Center).

Techniques: Concentration Assay, Derivative Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Autophagy-Mediated Inflammatory Cytokine Secretion in Sporadic ALS Patient iPSC-Derived Astrocytes

doi: 10.1155/2022/6483582

Figure Lengend Snippet: The company, Catalogue number and RRID of antibodies.

Article Snippet: Anti TRA-1-60 , Mouse , 1 : 200 , Abcam ab16288, RRID: AB_778563.

Techniques: