Journal: Cell reports

Article Title: Heterogeneity and transcriptional drivers of triple-negative breast cancer

doi: 10.1016/j.celrep.2023.113564

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: DU4475 cell line , ATCC , HTB-123.

Techniques: Recombinant, Mass Spectrometry, shRNA, Plasmid Preparation

Journal: bioRxiv

Article Title: Lead (Pb) exposure alters neural cell fate in the developing human brain

doi: 10.1101/2025.08.14.670354

Figure Lengend Snippet: a , Fetal and organoid-derived astrocytes and neurons were isolated by immunopanning using anti-HepaCAM and anti-CD24 antibodies, respectively. The fluorescent lead sensor leadmium was used to measure Pb uptake in purified cells. b , A representative leadmium trace from GW18 fetal astrocytes exposed to PbA-low (500 nM) then PbA-high (500 µM). Data is plotted as baseline-normalized leadmium fluorescence (DF/F) using a 20 min baseline. c , DF/F after 60-min exposure to Pb for fetal and organoid-derived cells. Each data point is the average of ∼8 cells from a single organoid differentiation or tissue sample (N=2 hiPSC lines and 3 organoid differentiations and N=3 fetal tissues). Error bars are mean and SD, statistical test is Brown-Forsythe ANOVA test and Dunnetts multiple comparison test. d , Brain tissue Pb-burdens from references – , respectively, are compared to cortical organoid Pb levels after exposure to Pb for 1-3 weeks. Each data point is a single literature value (left) or tissue measurement from a single organoid (right). ( d , inset) Pb measurements in organoid tissue show dose- and duration-dependent Pb increases even at maximal PbA-high (500 µM) exposures. e , GSEA analysis using Hallmark pathways for cortical organoids exposed to PbCl 2 for 1 week. f-h , Volcano plots highlighting specific differentially expressed genes related to ( f ) NRF2 pathway and metallothionines, ( g ) astrocyte markers, ( h ) glutathione cycle and pentose phosphate pathway. i , A schematic of the glutathione cycle and pentose phosphate pathway. j-k , Reduced glutathione (GSH) and oxidized (GSSG) glutathione was measured in whole organoid lysate by mass spectrometry. Each data point represents a single analytical sample from 3 pooled organoids. Antioxidant status is shown as the ratio of GSH/GSSG in j and the calculated redox potential in k . Error bars are mean and SD for all panels. The statistical comparison in ( j ) is Dunnett’s multiple comparisons test following a 1-way ANOVA.

Article Snippet: Astrocytes were isolated using mouse anti-HepaCAM (R&D systems, MAB4108), neurons were isolated using mouse anti CD24 (Miltenyi Biotec, 130-108-037) or mouse anti-Thy1 (BD Biosciences 550402), and radial glia were isolated using mouse anti-PTPRZ1 (Santa Cruz Biotechnology, sc-33664).

Techniques: Derivative Assay, Isolation, Purification, Fluorescence, Comparison, Mass Spectrometry, Analytical Sample Preparation

Journal: bioRxiv

Article Title: Lead (Pb) exposure alters neural cell fate in the developing human brain

doi: 10.1101/2025.08.14.670354

Figure Lengend Snippet: a , Radial glia were immunopanned (LIFR or PTPRZ1) from dissociated fetal cortices following depletion for CD24+ neurons and HepaCAM+ astrocytes. b , Representative images from dissociated cells with no immunopanning selection or immunopanned PTPRZ1 or LIFR cells. Cells were immunostained with PAX6 or HOPX (green) and GFAP (magenta) to quantify radial glia enrichment. Scale bar is 50 µm. c , 2D cultures of fetal radial glia were exposed to low doses on PbCl 2 -low or PbA-low for 3 days. EdU was added to quantify proliferation in culture (N=7-8 fetal samples) and live-dead imaging was used to quantify survival (N=3 fetal samples). Each point represents data from an individual fetal tissue sample. d , New-born cells were identified by EdU (magenta) and stained for SOX9 (green). Images shown are from a representative GW17 fetal sample. Scale bar is 50 µm. e , SOX9 immunoreactivity quantified for individual nuclei. The SOX9+ threshold is shaded in light green. Examples of nuclei across a spectrum of SOX9 intensity are shown in the right inset panels. f , Percent change in the %SOX9+ newborn cells relative to control for N=8-10 individual fetal samples. Data corresponding to the representative sample in e is highlighted in green.

Article Snippet: Astrocytes were isolated using mouse anti-HepaCAM (R&D systems, MAB4108), neurons were isolated using mouse anti CD24 (Miltenyi Biotec, 130-108-037) or mouse anti-Thy1 (BD Biosciences 550402), and radial glia were isolated using mouse anti-PTPRZ1 (Santa Cruz Biotechnology, sc-33664).

Techniques: Selection, Imaging, Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: The N-terminal Region of the Atypical Chemokine Receptor ACKR2 Is a Key Determinant of Ligand Binding *

doi: 10.1074/jbc.M113.534545

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Expression of ACKR2 was verified by flow cytometry using anti-ACKR2 monoclonal antibody and FITC-labeled secondary antibody (R&D Systems) or alternatively biotinylated anti-HA antibody (Miltenyi Biotec) and phycoerythrin-streptavidin-labeled secondary antibody (R&D Systems).

Techniques: Flow Cytometry, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: The N-terminal Region of the Atypical Chemokine Receptor ACKR2 Is a Key Determinant of Ligand Binding *

doi: 10.1074/jbc.M113.534545

Figure Lengend Snippet: Deletion of the tyrosine motif blocks ligand internalization by ACKR2. A , in mutant 1, all the tyrosines in the identified motif (labeled S to indicate likely sulfation) have been mutated to phenylalanine (labeled U to indicate likely unsulfation). Other than this, the sequences of WT ACKR2 and mutant 1 are identical. B , flow cytometric assessment of the expression levels of WT ACKR2 ( blue line ) and mutant 1 ( red line ) in stable transfected HEK cells. The proteins were measured using anti-HA antibodies to detect the HA tag at the extreme N terminus. Levels of expression are reflected in the extent of the right shift in the flow cytometry profile. Isotype control antibodies demonstrate the low level of nonspecific antibody staining ( green line ). C , flow cytometric profiles showing the internalization of Alexa-CCL2 by untransfected HEK cells ( panel i ), HEK cells expressing WT ACKR2 ( panel ii ), and HEK cells expressing mutant 1 ( Mut1 ; panel iii ). Note the prominent right shift in panel ii indicative of Alexa-CCL2 binding and uptake compared with the more limited right shift apparent in panel iii. D , summary of mean fluorescence intensity values obtained from binding/internalization of the indicated concentrations of Alexa-CCL2 by HEK cells expressing either WT ACKR2 or mutant 1. Note that untransfected HEK cells display a very low mean fluorescence intensity value indicative of low levels of nonspecific Alexa-CCL2 binding. The data were analyzed using one-way ANOVA with Tukey's post-test. E , Western blot analysis of CCL2 degradation over 24 h. Biotinylated CCL2 was incubated in the presence of HEK cells expressing either WT ACKR2 or mutant 1 (results from two clones shown), and the levels of intact CCL2 remaining were assessed using Western blotting and detection by streptavidin-HRP. Note the reduction in biotinylated CCL2 levels by wild type CCR2 but not by either of the two clones of mutant 1.

Article Snippet: Expression of ACKR2 was verified by flow cytometry using anti-ACKR2 monoclonal antibody and FITC-labeled secondary antibody (R&D Systems) or alternatively biotinylated anti-HA antibody (Miltenyi Biotec) and phycoerythrin-streptavidin-labeled secondary antibody (R&D Systems).

Techniques: Mutagenesis, Labeling, Expressing, Transfection, Flow Cytometry, Control, Staining, Binding Assay, Fluorescence, Western Blot, Incubation, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: The N-terminal Region of the Atypical Chemokine Receptor ACKR2 Is a Key Determinant of Ligand Binding *

doi: 10.1074/jbc.M113.534545

Figure Lengend Snippet: Inhibition of protein sulfation significantly inhibits ACKR2 activity. A , Western blot analysis of ACKR2 sulfation. Anti-HA beads were used to immunoprecipitate ACKR2 from a clone of HEK cells expressing WT ACKR2 and from a pool of HEK cells expressing mutant 1, as well as from two separately derived clones of mutant 1 transfectants. The 50-kDa molecular mass marker, which is coincident with migration of ACKR2 in SDS-PAGE gels, is shown in the upper panel . Expression was detected using an anti-sulfotyrosine antibody and loading normalized by reprobing the blot with an anti-HA antibody ( lower panel ). The 40- and 50-kDa molecular mass markers are indicated in the lower panel. B , mean fluorescence intensity values derived from flow cytometric assessment of Alexa-CCL22 binding to ACKR2-expressing HEK cells grown in either normal medium or medium supplemented with 30 m m sodium chlorate for the indicated times. The data were analyzed using one-way ANOVA with Tukey's post test. C , panel i , mean fluorescence intensity values measuring Alexa-CCL22 binding to ACKR2-expressing HEK cells treated with the indicated increasing concentrations of sodium chlorate. The graph also includes measurement of mean fluorescence intensity of Alexa-CCL22 binding to nontreated ACKR2 expressing HEK cells ( Non-treated ) and nontreated untransfected HEK cells ( Non-treated Neg ). Panel ii , flow cytometric profile demonstrating the reduction in Alexa-CCL22 binding by ACKR2-expressing HEK cells before ( blue line ) and after ( red line ) treatment for 6 h with 150 m m sodium chlorate. Panel iii , Western blot analysis of lysates of cells expressing ACKR2 using anti-sulfotyrosine antibodies. Cells were either left untreated or treated for 6 days with 150 m m sodium chlorate. The 40- and 50-kDa molecular mass markers are indicated. Gel loading was normalized by reprobing with antibodies to β-actin ( lower panel ).

Article Snippet: Expression of ACKR2 was verified by flow cytometry using anti-ACKR2 monoclonal antibody and FITC-labeled secondary antibody (R&D Systems) or alternatively biotinylated anti-HA antibody (Miltenyi Biotec) and phycoerythrin-streptavidin-labeled secondary antibody (R&D Systems).

Techniques: Inhibition, Activity Assay, Western Blot, Expressing, Mutagenesis, Derivative Assay, Clone Assay, Marker, Migration, SDS Page, Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The N-terminal Region of the Atypical Chemokine Receptor ACKR2 Is a Key Determinant of Ligand Binding *

doi: 10.1074/jbc.M113.534545

Figure Lengend Snippet: Individual tyrosine residues do not account for the ligand internalizing activity of ACKR2. A , flow cytometric assessment, using anti-HA antibodies, of the expression levels of mutants 2–5 in the transfected HEK cells. Note that the extent of the right shift of the flow cytometric profile is indicative of the extent of expression of the mutants. B , summary of mean fluorescence intensity values obtained from flow cytometric analysis of the binding of Alexa-CCL2 to WT ACKR2 and mutants 1–4. Note again that untransfected HEK cells do not significantly bind Alexa-CCL2. C , summary of mean fluorescence intensity values obtained from flow cytometric analysis of the binding of Alexa-CCL22 to mutant 5 compared with untransfected HEK cells and WT ACKR2-expressing HEK cells. D , flow cytometric assessment using anti-HA antibodies of the expression levels of mutants 1 and 6–9 in the transfected HEK cells. E , summary of mean fluorescence intensity values obtained from flow cytometric analysis of the binding of Alexa-CCL22 to WT ACKR2, mutant 1, and mutants 6–9 all expressed in HEK cells. Note again that untransfected HEK cells do not significantly bind Alexa-CCL22. All data were analyzed using one-way ANOVA with Tukey's post test.

Article Snippet: Expression of ACKR2 was verified by flow cytometry using anti-ACKR2 monoclonal antibody and FITC-labeled secondary antibody (R&D Systems) or alternatively biotinylated anti-HA antibody (Miltenyi Biotec) and phycoerythrin-streptavidin-labeled secondary antibody (R&D Systems).

Techniques: Activity Assay, Expressing, Transfection, Fluorescence, Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The N-terminal Region of the Atypical Chemokine Receptor ACKR2 Is a Key Determinant of Ligand Binding *

doi: 10.1074/jbc.M113.534545

Figure Lengend Snippet: A sulfated peptide derived from the ACKR2 N terminus binds ligand. A , sequences of peptides designed from the N terminus of ACKR2 and generated by peptide synthesis. SUL* indicates a sulfatable tyrosine residue. ACKR2-N(s) represents the sequence of the fully sulfated peptide whereas ACKR2-N(un-s) indicates a fully unsulfated peptide. The hexahistidine tags are indicated at the extreme C terminus of the peptide. B , mass spectrometry analysis of the mixed levels of sulfation apparent in the sulfated version of ACKR2-N. The mass/charge ( m / z ) profile is shown in panel i , and the deconvoluted molecular mass profile is shown in panel ii . Prominent m / z peaks are indicated in panel i , and the molecular mass peaks corresponding to the differentially sulfated peptide variants are indicated in panel ii . In panel ii , the extent of sulfation of the different variants is indicated in parentheses after the molecular mass. C , Western blot analysis ( panel i ) using an anti-ACKR2 antibody and subsequent densitometry analysis ( panel ii ) indicate the presence of ACKR2-N(s) after pulldown binding assays using biotinylated-CCL2. As shown, the ACKR2 peptide migrates as two bands of 8 and 14 kDa. Note the markedly higher pulldown of the ACKR2 peptide by biotinylated CCL2 ( lane 1 ) compared with biotinylated CCL19 ( lane 2 ). The pulldown achieved by biotinylated CCL19 is not significantly different from the background binding of ACKR2-N(s) to the streptavidin beads used for the pulldown ( lane 3 ). Note also the lack of nonspecific antibody cross-reacting with 4 μg of CCL2, confirming the specificity of the antibody in this assay. D , addition of sulfated ACKR2 peptide (ACKR2-N(s)), but not the unsulfated ACKR2 peptide (ACKR2-N(un-s)), significantly reduced uptake of Alexa-CCL2 by transfected HEK ACKR2 cells, as determined by flow cytometric analysis (data are reported as mean fluorescence intensity values from the flow cytometric analysis). The data were analyzed using one-way ANOVA with Tukey's post test. E , addition of ACKR2-N(s) significantly reduced uptake of Alexa-CCL2 by CCR2 expressed on THP1 cells, as determined by flow cytometric analysis. AF-CCL2 only refers to THP1 cells exposed to Alexa-CCL2, and AF-CCL2 + ACKR2-N(s) refers to THP1 cells exposed to Alexa-CCL2 as well as the sulfated N-terminal peptide. The data were analyzed using Student's t test.

Article Snippet: Expression of ACKR2 was verified by flow cytometry using anti-ACKR2 monoclonal antibody and FITC-labeled secondary antibody (R&D Systems) or alternatively biotinylated anti-HA antibody (Miltenyi Biotec) and phycoerythrin-streptavidin-labeled secondary antibody (R&D Systems).

Techniques: Derivative Assay, Generated, Residue, Sequencing, Mass Spectrometry, Western Blot, Binding Assay, Transfection, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The N-terminal Region of the Atypical Chemokine Receptor ACKR2 Is a Key Determinant of Ligand Binding *

doi: 10.1074/jbc.M113.534545

Figure Lengend Snippet: The serine protease staphopain A cleaves ACKR2 at the N terminus. A , ACKR2 expressed on HEK cells is cleaved by staphopain A ( Staph A ) as determined by Western blot analysis using an antibody specific for the C terminus of ACKR2. In this experiment β-tubulin was used as a loading control. Full-length (*) and truncated (** and ***) ACKR2 proteins are indicated. ACKR2-expressing HEK cells were treated either with PBS or with staphopain A for the indicated time points. Untransfected HEK cells treated with staphopain A are included as a negative control. The gel positions corresponding to 40, 50, and 60 kDa are indicated. B , densitometric analysis of band intensity from the blot shown in A. NEG refers to the PBS-treated ACKR2-transfected HEK cells. In this figure the full-length ACKR2 is marked with *, and the truncated ACKR2 is marked with *** in A . Expression levels are normalized to the β-actin loading control. C , flow cytometric assessment of the effects of staphopain A on Alexa-CCL22 internalization by ACKR2-expressing HEK cells. The data are presented as mean fluorescence intensity values measured from flow cytometric analysis of Alexa-CCL22 binding. HEK-AF-CCL22 represents the levels of Alexa-CCL22 binding to untransfected HEK cells. ACKR2-PBS represents the background fluorescence of ACKR2 transfected HEK cells. ACKR2-AF-CCL22 represents the binding of Alexa-CCL22 to ACKR2-transfected HEK cells. ACKR2-AF-CCL22/Staph. A (2m m ) represents cells pretreated with 2 μ m staphopain A prior to Alexa-CCL22 binding. Note, in this experiment, that the alternative ACKR2 ligand, CCL22, is used in place of CCL2. The data were analyzed using one-way ANOVA with Tukey's post test. D , panel i , flow cytometric profiles showing the expression of ACKR2 in untransfected ( upper panel ) and transfected ( lower panel ) CHO cells. ACKR2 expression levels were measured using anti-HA antibodies. The right shift of the flow cytometric profile for CHO-ACKR2 cells indicates high level expression. Panel ii , measurement of Alexa-CCL22 ( AF-CCL22 ) binding to ACKR2-expressing CHO cells in the presence, or absence, of increasing concentrations of staphopain A. The data were obtained by measuring fluorescence intensity in a standard fluorescent plate-reader. Background fluorescence is shown by the data labeled untreated . The data presented in this figure are representative of more than two repeat experiments.

Article Snippet: Expression of ACKR2 was verified by flow cytometry using anti-ACKR2 monoclonal antibody and FITC-labeled secondary antibody (R&D Systems) or alternatively biotinylated anti-HA antibody (Miltenyi Biotec) and phycoerythrin-streptavidin-labeled secondary antibody (R&D Systems).

Techniques: Western Blot, Control, Expressing, Negative Control, Transfection, Fluorescence, Binding Assay, Labeling

Journal: Scientific Reports

Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

doi: 10.1038/s41598-017-17951-5

Figure Lengend Snippet: Characterization of the single-chain version of the coat protein dimer containing the His-tag. ( A ) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), molecular weight of wild type MS2 bacteriophage coat protein was about 13 kDa (A1) whereas single chain version of the MS2 coat protein dimer containing the His-tag was about 28 kDa (A2); ( B ) western blot analysis using primary Anti-Enterobacterio Phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) and secondary goat anti-rabbit IgG conjugated with HRP (Jackson ImmunoResearch, UK) antibodies at wild type MS2 bacteriophage coat protein (B1) and single chain version of the MS2 coat protein dimer containing the His-tag (B2); ( C ) western blot analysis using primary anti-HisTag antibody (Pierce, Thermo Scientific, USA) and secondary goat anti-mouse IgG conjugated with HRP (Jackson ImmunoResearch) antibodies at wild type MS2 bacteriophage coat protein (C1) and single chain version of the MS2 coat protein dimer containing the His-tag (C2); ( D , E ) laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) analysis of wild-type bacteriophage MS2 coat protein and single-chain version of the MS2 coat protein dimer containing the His-tag; M, marker, spectra multicolor broad range protein ladder (Fermentas); the marker values are in kDa. The figures were cropped, full length gel and blots are presented in Supplementary Figures , and .

Article Snippet: Western blotting with an anti-HisTag antibody (Pierce, Thermo Scientific, USA) diluted 1:3000 as primary antibody and goat anti-mouse IgG conjugated with HRP diluted 1:1000 (Jackson ImmunoResearch) as a secondary antibody was performed as described above.

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Molecular Weight, Western Blot, Mass Spectrometry, Marker