cxcl12  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Adenovirus E4ORF1 activates isoform-specific phosphatidylinositol 3-kinase signaling in human endothelial cells

doi: 10.1016/j.jbc.2025.110947

Figure Lengend Snippet: Human Ad5E4ORF1 associates with scaffold proteins IQGAP1, DLG1, CASK, and LIN7C but only DLG1 promotes E4ORF1-mediated AKT activation . A , western blot analysis of Ad5E4ORF1 proteins and phosphorylated AKT and ERK1/2 in HUVECs. ( Top panel ) Cells infected with vector control or Ad5E4ORF1 viruses were starved for 4 h prior to cell lysis in LDS loading buffer. Tagged and untagged Ad5E4ORF1 proteins were probed with Ad5E4ORF1-, Flag-, or HA-specific antibodies. VCN ( bottom ), genomic lentiviral vector copy numbers. ( Lower panels ) Densitometry quantification of immunoblots shown in the top panel . Phosphorylated AKT and ERK signals were normalized to their respective total protein levels and expressed relative to the vector control, which was set to 1. Ad5E4ORF1 protein levels, normalized to GAPDH, were quantified from anti-Ad5E4ORF1 immunoblots and expressed relative to native (untagged) E4ORF1. Data are presented as mean ± SD from 3 to 5 independent HUVEC lines. Statistical analysis was performed using one-way ANOVA (two-sided), followed by Tukey’s post hoc test. ns, nonsignificant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001 ( p value notation used throughout all figures). B , Ad5E4ORF1-enabled cell survival under starvation. Indicated transduced HUVECs were kept in minimal X-Vivo 20 medium for 7 days and cell survival rates (percentage of live cells in starvation relative to those in growth medium) presented as mean ± SD. One-way ANOVA with Tukey’s test (n = 3 independent HUVEC lines). C , identification of Ad5E4ORF1-interacting proteins in HUVECs. Cells expressing GFP (−) or Flag-Ad5E4ORF1 (+) were subjected to cross-linking anti-Flag immunoprecipitation. Flag peptide-eluted proteins were resolved on SDS-PAGE and gel slices analyzed by mass spectrometric protein ID. Shown is an SDS-PAGE silver stain with major identified candidate proteins marked on the right. The band corresponding to LIN7C was identified based on increased signal intensity over a comigrating background protein, denoted by an asterisk (∗). D , confirmation of identified interactions by protein immunoprecipitation (IP) in HUVECs expressing HA-tagged wild type (WT) or mutant Ad5E4ORF1. ΔC3, deletion of C terminal 3 amino acids (part of PDZ-binding motif). Whole cell lysates were prepared in IGEPAL CA-630 buffer without cell cross-linking. Bead-bound proteins, along with 15% of the corresponding input lysates, were analyzed by western blot. Data shown represent experiments using three HUVEC lines. E , interdependence of Ad5E4ORF1 scaffold interactions. DLG1, CASK, or LIN7C gene-specific shRNAs or a nontargeting (NT) shRNA were stably expressed under doxycycline induction for 4 days in control and HA-Ad5E4ORF1-transduced HUVECs. ( Left panel ) Immunoprecipitation blot performed as in ( D ). ( Right panels ) Densitometry quantification of DLG1, CASK, and LIN7C IP efficiency (IP/Input) in gene knockdown (KD) HUVECs relative to NT control. One-way ANOVA with Tukey’s test (mean ± SD; n = 3 HUVEC lines). F , schematic model of Ad5E4ORF1 interactions with scaffold proteins. G and H , effect of scaffold protein knockdowns on AKT activation. Indicated gene-specific shRNAs were doxycycline-induced for 4 days in HUVECs, which were then starved for 4 h and subjected to western blot analysis. Data shown represent experiments using three HUVEC lines. ( H , lower panel ) Quantification of AKT phosphorylation (average of two shRNAs) from upper panel blots. One-way ANOVA with Tukey’s test (mean ± SD; n = 3 HUVEC lines). I , effect of DLG1 knockdown on receptor-mediated AKT activation. DLG1-specific (+) and nontargeting (NT) shRNAs were doxycycline-induced in HUVECs for 4 days. After 4 h of starvation, cells were stimulated for 5 min with the indicated cytokines (20 ng/ml FGF2, HGF, SDF1; or 100 nM S1P) and lysed directly into LDS loading buffer for western blot. ( Lower panels ) Quantification of AKT and ERK phosphorylation from upper panel blots. Paired t test (mean ± SD; n = 3 HUVEC lines). Ad5E4ORF1, human adenovirus serotype 5 early gene E4ORF1; AKT, protein kinase B; CASK, calcium/calmodulin-dependent serine protein kinase; DLG1, discs large homolg 1; ERK, extracellular signal-regulated kinase; FGF2, fibroblast growth factor 2; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cells; IQGAP1, IQ motif containing GTPase activating protein 1; LDS, lithium dodecyl sulfate; LIN7C, Lin-7 Cell Polarity Scaffold C; SDF1, stromal cell-derived factor-1; VCN, viral copy number.

Article Snippet: Other cytokines: HGF (PeproTech 100–39H); SDF1 (R&D Systems 350-NS); and S1P (Sigma-Aldrich 73914).

Techniques: Activation Assay, Western Blot, Infection, Plasmid Preparation, Control, Lysis, Expressing, Immunoprecipitation, SDS Page, Silver Staining, Mutagenesis, Binding Assay, shRNA, Stable Transfection, Knockdown, Phospho-proteomics, Derivative Assay

Journal: bioRxiv

Article Title: IgE-producing cells on the move: CCR2 is a key regulator of IgE + plasma cell migration

doi: 10.64898/2025.12.18.695109

Figure Lengend Snippet: (A) Switched tonsil B cells were stained for surface CXCR4 followed by intracellular for IgE and IgG1. The histogram shows CXCR4 expression on IgE + (red) and IgG1 + (blue) gated cells. The filled grey histogram represents the isotype control staining. The accompanying graph shows CXCR4 median fluorescence intensity (MFI) on the surface of IgE + and IgG1 + cells. (B) CXCL12 induced migration of IgE + and IgG1 + cells was assessed using the transwell assay. After 3h of migration, the number of migrating cells was quantified by flow cytometry. Migration of IgE + and IgG1 + cells in response to 300 ng/mL (30nM) of CXCL12 is shown as a percentage of the cells migrating in response to RPMI control. (C) Flow cytometry dot plots of the IgE + and IgG1 + gated GC-like B cells, a PC-like “plasmablast” and PCs. Representative histograms show the CXCR4 expression on each of these gated IgE + and IgG1 + cells. (D) Bar chart showing the CXCR4 expression (MFI) across different IgE + and IgG1 + cell populations. (E) Flow cytometry staining of IgE + and IgG1 + GC-like B cells, PC-like PBs and PC after 3h of migration to the bottom chamber of the transwell. (F) CXCL12 induced migration shown as a percentage of the cells migrating in response to RPMI control. Data are mean + s.d. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test (D, F) or paired two-tailed t -test with Welch’s correction (A, B); *p< 0.05; **p< 0.01; ***p< 0.001; and ****p< 0.0001. Non-significant values are not shown.

Article Snippet: More specifically, we tested the migration of IgE + and IgG1 + cells in response to recombinant human CXCL12 (R&D systems; 300ng/mL), CCL2 (Biolegend; 10ng/mL, 100ng/mL and 300ng/mL) and CCL28 (R&D systems; 300ng/mL and 1.5ug/mL).

Techniques: Staining, Expressing, Control, Fluorescence, Migration, Transwell Assay, Flow Cytometry, Comparison, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: ARAP1 fine-tunes F-actin polymerization level in lymphocytes through RhoA inhibition

doi: 10.3389/fimmu.2025.1591450

Figure Lengend Snippet: ARAP1 localization in lymphocytes after chemokine stimulation. (A) ARAP1 protein expression in Ba/F3.hLFA1 cells following ARAP1 knockout (KO). (B) CXCR4 expression of wild-type (WT) cells (red) and Arap1 knockout (KO) cells (light blue). Unstained control WT cells (dotted red) and Arap1 KO cells (dotted light blue) were also shown. (C) Integrin αL expression in WT cells (red) and Arap1 KO cells (light blue), measured using flow cytometry and TS2/4 antibody staining. Unstained control WT cells (dotted red) and Arap1 KO cells (dotted light blue) were also shown. (D) Representative Z-stack images of endogenous ARAP1 in cells unstimulated and stimulated with CXCL12 (10 nM, 1 and 10 min). ARAP1, green; F-actin: red. Arrow heads indicate ARAP1 accumulation in ruffle membranes. Scale Bar, 5 μm. (E) Representative Z-projection images of Venus-ARAP1 wild type in the enter body of cells unstimulated and stimulated with CXCL12 (10 nM, 1 and 10 min). ARAP1, green; F-actin, red. Scale Bar, 10 μm. Arrowheads indicate colocalization of Venus-ARAP1 and F-actin rich ruffles.

Article Snippet: Recombinant CXCL12 (350-NS) and CCL21 were purchased from R&D Systems.

Techniques: Expressing, Knock-Out, Control, Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: ARAP1 fine-tunes F-actin polymerization level in lymphocytes through RhoA inhibition

doi: 10.3389/fimmu.2025.1591450

Figure Lengend Snippet: ARAP1 deficiency increased F-actin capping and polymerization in lymphocytes. (A) Quantification of F-actin level in WT (n = 3) and Arap1 KO (n = 3) cells following stimulation with CXCL12 (10 nM). F-actin level was visualized by staining with fluorescently labeled phalloidin and measured using flow cytometry. Median fluorescent intensity (MFI) was normalized to the average MFI of unstimulated WT cells. (B) The upper representative image showed F-actin and CD44 staining in unpolarized cells and chemokine-induced polarized cells (10 nM, 10 min), acquired using imaging cytometer. The lower panel shows average percentages of the F-actin capped cells among WT (n = 3) and Arap1 KO (n = 3) cells before and after CXCL12 stimulation (1, 10, 50 nM, 10 min). (C) Representative Z-projection images of WT and Arap1 KO cells stained with fluorescently labeled phalloidin. Scale Bar, 10 μm. Asterisks indicate statistical significance for (A, B) calculated using Student’s t -test; * p < 0.05; ** p < 0.01.

Article Snippet: Recombinant CXCL12 (350-NS) and CCL21 were purchased from R&D Systems.

Techniques: Staining, Labeling, Flow Cytometry, Imaging, Cytometry

Journal: Frontiers in Immunology

Article Title: ARAP1 fine-tunes F-actin polymerization level in lymphocytes through RhoA inhibition

doi: 10.3389/fimmu.2025.1591450

Figure Lengend Snippet: Ectopic ARAP1 expression inhibits RhoA activation via RhoGAP and the RA domain. (A) Schematic diagram of ARAP1 mutants with RhoGAP domain (ΔRhoGAP) or RA domain (ΔRA) deletion. (B) Expression of Venus alone, Venus-ARAP1 wild-type (WT), Venus-ARAP1ΔRhoGAP, and Venus-ARAP1ΔRA in Ba/F3 transfectants, detected using western blotting. The arrowhead shows endogenous ARAP1. (C) Detection of active RhoA in cells expressing Venus alone or Venus-Arap1 mutants using GST-Rhotekin-RBD pull-down assay. Both active (GTP-bound, the upper image) and total RhoA (the lower image) were detected using anti-RhoA antibody. The numbers under images represent relative active RhoA expression calculated by dividing the intensity of RhoA-GTP by that of total RhoA and normalizing the calculated ratios to that of unstimulated Venus-transfected cells. (D) RhoA activation levels in Ba/F3 transfectants expressing Venus alone (n = 3), Venus-ARAP1WT (n = 3), Venus-ARAP1ΔRhoGAP (n = 3), and Venus-ARAP1ΔRA (n = 3) mutants with and without CXCL12 stimulation (10 nM, 5 min) measured using flowcytometry with anti-RhoA-GTP and anti-total RhoA staining. RhoA activation levels were calculated as the intensity of RhoA-GTP staining divided by intensity of total RhoA staining. The calculated ratios were then normalized to the average ratio of unstimulated Venus-transfected cells. (E) Quantification of F-actin level in cells expressing Venus (n = 3), Venus-ARAP1WT (n = 3), and Venus-ARAP1ΔRhoGAP (n = 3) mutant following stimulation with CXCL12 (10 nM) using fluorescently labeled phalloidin staining. Percentages of polarized cells are shown. (F) Confocal imaging of cells expressing ARAP1 mutants stained with fluorescently labeled phalloidin. Scale bars, 10 µm. Statistical analysis for (D, E) was performed using one-way ANOVA with Tukey’s multiple comparison test. Asterisks indicate statistical significance; * p < 0.05, ** p < 0.01.

Article Snippet: Recombinant CXCL12 (350-NS) and CCL21 were purchased from R&D Systems.

Techniques: Expressing, Activation Assay, Western Blot, Pull Down Assay, Transfection, Staining, Mutagenesis, Labeling, Imaging, Comparison

Journal: Frontiers in Immunology

Article Title: ARAP1 fine-tunes F-actin polymerization level in lymphocytes through RhoA inhibition

doi: 10.3389/fimmu.2025.1591450

Figure Lengend Snippet: ARAP1-RA domain bound to Rap1. (A) Co-immunoprecipitation assay of Venus-ARAP1 and SNAP-Rap1 using anti-GFP antibody. The left panel shows precipitated Venus alone and Venus-Arap1; the middle panel shows co-immunoprecipitated SNAP alone and SNAP-Rap1; the right panel shows the inputs of SNAP and SNAP-Rap1 from whole lysates. (B) The distribution of Venus-ARAP1 and Turquoise2-Rap1 in unstimulated and CXCL12-stimulated (CXCL12, 10 nM, 1 min) cells. Confocal images of Venus-ARAP1 and Turquoise2-Rap1 and the merged images are shown. Arrowheads represent the location where ARAP1 and Rap1 colocalize. Scale bar, 10 µm (C) Schematic diagram of GST, GST-RA, and GST-RA-PH5-C-terminal domains. The image shows Coomassie staining of these proteins enriched using Glutathione Sepharose beads. (D) RA domain of ARAP1 bound to Rap1. Lysates from 293T cells transfected with SNAP-Rap1 mutants were subjected to pull-down assay using GST, GST-RA, and GST-RA-PH5-C-terminal domain. Representative images of pull-down samples (upper image) and their inputs (lower image) are shown.

Article Snippet: Recombinant CXCL12 (350-NS) and CCL21 were purchased from R&D Systems.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Staining, Transfection, Pull Down Assay

Journal: Frontiers in Immunology

Article Title: ARAP1 fine-tunes F-actin polymerization level in lymphocytes through RhoA inhibition

doi: 10.3389/fimmu.2025.1591450

Figure Lengend Snippet: ARAP1 inhibited lymphocyte migration. (A) Chemotactic migration efficiency of WT and Arap1KO T cells. Cells (1 × 10 5 ) were loaded onto upper chamber and allowed to migrate to lower chamber for 3 h The lower chambers contained CXCL12 at concentrations of 0 (n = 5), 10 nM (n = 5). (B) Chemotactic migration efficiency of WT and Arap1KO T cells on integrin-ligand ICAM-1. The upper chambers were coated with 100 μg/ml of anti-human IgG capture antibody, followed by loading of 0 or 0.5 μg/ml of human ICAM1. The lower chambers contained CXCL12 at concentrations of 0 (n = 3), 10 nM (n = 3). (C) Migration efficiency of Ba/F3.hLFA1 cells expressing ARAP1 mutants towards CXCL12 using transwell migration assay. The lower chambers contained CXCL12 at concentrations of 0 (n = 1), 3 nM (n = 3). (D) Migration of Ba/F3.hLFA1 cells expressing ARAP1 mutants on immobilized ICAM-1 (0.5 μg/ml) and CXCL12 (50 nM). Upper images display individual cell tracks. Scale bar, 50 µm. The lower left panel showed track velocity (μm/min); the lower right panel confinement ratio calculated as the displacement divided by total path length. Statistical analysis was performed using Student’s t -test for (A, B) and using one-way ANOVA with Tukey’s multiple comparison test for (C, D) Asterisks indicate statistical significance; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant CXCL12 (350-NS) and CCL21 were purchased from R&D Systems.

Techniques: Migration, Expressing, Transwell Migration Assay, Comparison

Journal: Bone Research

Article Title: Neonatal bone marrow interstitial fluid supports expansion and osteogenic ability of human bone marrow mesenchymal stromal cells

doi: 10.1038/s41413-025-00496-z

Figure Lengend Snippet: NBIF promotes CXCR4 expression and enhances hBMSC homing to bone marrow. a, b Quantitative RT-PCR analysis of CXCR4 expression in hBMSCs after 7-day culture ( a ) and in mouse primary mBMSCs after 2 passages (7 days) ( b ). Data [and also in ( d )] were expressed as mean ± standard error of the mean (SEM) of the fold change across three replicates for each group. P -values were obtained from an unpaired t -test; ** P ≤ 0.01, **** P ≤ 0.000 1. c, d Representative images ( c ) and quantification data ( d ) of migratory hBMSCs in the Transwell culture (see the Materials and Methods section for details). rhCXCL12, recombinant human CXCL12 protein; AMD3100, the CXCR4 antagonist. Scale bar, 100 μm. e Scheme of the experimental design for mouse transplantation and analysis of GFP-labeled hBMSCs. f Quantification of the proportion of GFP + -hBMSCs in the whole bone marrow of host mice. Data were expressed as mean ± standard error of the mean (SEM) across indicated replicates for each group. 5 mice for the FBS-fed hBMSCs group and 5 mice for NBIF-fed hBMSCs group at each time point. P -values were obtained from an unpaired t -test; * P < 0.05, ** P < 0.01. g Representative immunofluorescent images of markers at 14 days post-transplantation. Scale bar, 10 μm. h–j Quantification of GFP + - ( h ), LEPR + - ( i ) or LEPR + ; GFP + - cells ( j ) in FBS-fed hBMSCs group ( n = 5 mice) or NBIF-fed hBMSC group ( n = 10 mice) 14 days post transplantation. Data were expressed as mean ± standard error of the mean (SEM) for each group. P -values were obtained from an unpaired t -test; * P < 0.05, ** P < 0.01

Article Snippet: To assess the effect of CXCL12, recombinant human CXCL12 was added to the lower chamber at a concentration of 100 ng/mL, with or without 50 nmol/L CXCR4 inhibitor AMD3100 (Medchemexpress) for 24 hours.

Techniques: Expressing, Quantitative RT-PCR, Recombinant, Transplantation Assay, Labeling