anti human e selectin ab  (R&D Systems)

Journal: bioRxiv

Article Title: CA19-9 promotes liver metastasis of pancreatic cancer through E-selectin mediated extravasation

doi: 10.64898/2026.04.08.717301

Figure Lengend Snippet: (A) Representative images of adhesion assay using Calcein-AM stained CA19-9 negative (top) and positive (bottom) cells adhered to HUVECs. Scale bars = 100 μm. (B) Quantification of adhered CA19-9 neg and CA19-9 pos FC1199 (left), KPCY (middle) and FC1245 (right) cells per field. (C, D) Quantification of adhered CA19-9 pos FC1199 cells treated with anti-CA19-9 antibody (5B1; αCA19-9) (C) and anti-human E-selectin antibody (BBA16; αSELE) (D) compared with isotype control (ISO). (E, F) Quantification of adhered hM19a 2D cells treated with anti-CA19-9 antibody (5B1; αCA19-9) (E) and anti-human E-selectin antibody (BBA16; αSELE) (F) compared with isotype control (ISO). (G) Quantification of adhered Capan-2 cells after FUT3 knockout (sgFUT3) compared with a negative control (sgCtrl). *Data are presented as mean ± SD. Data are representative of at least two independent experiments. Statistical significance was determined by unpaired two-tailed t-test with Welch’s correction. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Alternatively, HUVECs were incubated with mouse IgG (Leinco Technologies, Inc., I-536) or anti-human E-selectin Ab (R&D Systems, BBA16) at 25 μg/mL for 30 minutes at 37°C.

Techniques: Cell Adhesion Assay, Staining, Control, Knock-Out, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: CA19-9 promotes liver metastasis of pancreatic cancer through E-selectin mediated extravasation

doi: 10.64898/2026.04.08.717301

Figure Lengend Snippet: (A) Schema of study design for metastatic seeding analysis in (B, C). (B) Representative YFP IHC of liver sections one day after splenic injection of CA19-9 neg or CA19-9 pos KPCY cells. Scale bar = 200 μm. (C) Quantification of YFP+ tumor cells in liver sections of WT mice injected with CA19-9 neg or CA19-9 pos KPCY cells, normalized by tissue area (mm²). (D) Schema of study design in (E-H). (E) Representative macroscopic images of livers isolated from WT (left) and E-selectin KO (right) mice injected with CA19-9 pos cells. Scale bar = 1 cm. (F, G) Quantification of liver weight (F) and of liver weight normalized by body weight (G). (H) Quantification of CK19 positive area (%) across the whole liver sections. (I) Schema of study design in (J) (J) Quantification of YFP+ tumor cells in liver sections of WT and E-selectin KO mice injected with CA19-9 pos KPCY cells, normalized by tissue area (mm²). (K) Schema of study design in (L, M) (L, M) Quantification of CA19-9 pos KPCY cells in liver sections of WT mice injected with CA19-9 pos KPCY cells and treated with isotype control or anti-CA19-9 antibody (5B1; αCA19-9) (L), or isotype control or anti-mouse E-selectin antibody (9A9; αSele) (M), normalized by tissue area (mm²). *Data are presented as mean ± SD. Each dot represents an individual mouse. For A-H, mice were injected with 1 × 10 5 tumor cells per mouse. For I-M, mice were injected with 3 × 10 5 tumor cells per mouse. Statistical significance was calculated using unpaired two-tailed t-test with Welch’s correction. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Alternatively, HUVECs were incubated with mouse IgG (Leinco Technologies, Inc., I-536) or anti-human E-selectin Ab (R&D Systems, BBA16) at 25 μg/mL for 30 minutes at 37°C.

Techniques: Injection, Isolation, Control, Two Tailed Test

Journal: The FASEB Journal

Article Title: E‐selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity

doi: 10.1096/fj.202000662rr

Figure Lengend Snippet: FIGURE 1 shRNA infection reduced E-selectin expression and enhanced transendothelial migration (TEM) of polymorphonuclear neutrophils (PMNs) on human umbilical vein endothelial cell (HUVEC) monolayer. A, mRNA expressions of E- and P-selectins, ICAM-1, and VCAM-1, on 4 h-LPS treated, scr or shE-sel HUVECs. Data were obtained by qPCR and normalized to the scr cells. E-selectin expression decreased in HUVEC monolayer examined by (B) flow cytometry analysis, (C) western blotting, and (D) immunostaining. Cells incubated with isotype-matched Abs were used as control in flow cytometry and immunostaining. E, Time courses of PMN TEM ratio on shE-sel, scr, or intact HUVEC monolayer. Data were obtained from at least three repeats and presented as the mean ± SE (shadow bands). *P < .05, and #P < .05, compared with either scr or intact controls, respectively. F, Typical images of an in vitro PMN TEM assay in three cases. White arrows indicated the individual PMNs that are undergoing TEM

Article Snippet: Basic fibroblast growth factor (bFGF), rabbit anti- VE- cadherin phospho- specific [Tyr731] (NBP254766), and anti- human E- selectin (NBP1- 45545) Abs were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: shRNA, Infection, Expressing, Migration, Flow Cytometry, Western Blot, Immunostaining, Incubation, Control, In Vitro

Journal: The FASEB Journal

Article Title: E‐selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity

doi: 10.1096/fj.202000662rr

Figure Lengend Snippet: FIGURE 2 E-selectin knockdown lowered the integrity of human umbilical vein endothelial cell (HUVEC) monolayer. A, Localization of vascular endothelial (VE)-cadherin and F-actin from immunofluorescence staining. The gap between two neighboring cells was defined as previously reported.51Red arrows indicated the gaps at cell-cell junctions. B, F-actin anisotropy estimated for scr (n = 75) and shE-sel (n = 54) cells. For definition, see the Materials and Methods. C, Global or local elastic moduli of scr or shE-sel HUVEC monolayer. Height (first column), topography (second column), and modulus (third column) images of scr or shE-sel HUVEC monolayer were obtained using PFQNM-LC probes in QNM mode. Individual cell was segregated into two regions by plotting an arbitrary line in the height image (white lines in first column) and defining the cell body as the region with height ≥1 μm and the cell periphery as the one with height <1 μm (red lines in fourth column). Each panel is 100 × 100 μm2. D, Quantification of inter-endothelial gap areas for scr (n = 318) or shE-sel (n = 389) gaps from 90 to 100 interconnected cells. E, Regional elastic moduli of cell body and cell periphery. Data were collected from a total of 36–46 randomly selected regions of the acquired images (see the Materials and Methods) and presented as the mean ± SE. F, Typical vascular endothelial (VE)-cadherin staining for scr and shE-sel HUVEC monolayers. G, Representative patches used for manual classification of adherence junctions in three categories: straight, fingers, and reticular junctions. H, Morphological analysis of VE-cadherin-labeled patches on scr (n = 441) or shE-sel (n = 428) cells. Data in (B), (D), (E), and (H) were presented as the mean ± SE from at least three repeats and compared using a t test. *P < .05, ***P < .001, ****P < .0001

Article Snippet: Basic fibroblast growth factor (bFGF), rabbit anti- VE- cadherin phospho- specific [Tyr731] (NBP254766), and anti- human E- selectin (NBP1- 45545) Abs were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Immunofluorescence, Staining, Labeling

Journal: The FASEB Journal

Article Title: E‐selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity

doi: 10.1096/fj.202000662rr

Figure Lengend Snippet: FIGURE 3 E-selectin regulated PMN transmigration via Arp2/3 complex. Effects of Arp2/3 inhibition on the transendothelial migration (TEM) of polymorphonuclear neutrophils (PMNs) across scr (A) or shE-sel (B) human umbilical vein endothelial cell (HUVEC) monolayer. Data were obtained from at least three repeats and presented as the mean ± SE (shadow bands). #P < .05 and *P < .05, compared with CK689 and dimethyl sulfoxide (DMSO) controls, respectively. Effects of Arp2/3 inhibition (C, D) in elastic moduli of intact HUVECs at cell–cell junction. Data were collected from a total of 31-43 randomly selected regions of the acquired images (see the Materials and Methods) and presented as the mean ± SE in D. (E, F, G) F-actin distribution and junctional gap presence in Arp2/3-inhibited HUVEC monolayer. F-actin anisotropy was estimated for DMSO (n = 70), CK689 (n = 63), and CK666 (n = 56) cases (F). Red arrows indicated the gaps at cell–cell junctions (E), and total 527-700 gaps of 90-100 interconnected HUVEC cells were collected (G). Regulation of Arp2/3 complex in the formation of adherence junctions (H, I). Morphological analysis of vascular endothelial (VE)-cadherin-labeled patches by CK666 (n = 324), CK689 (n = 380), and DMSO (n = 356) treatment (I). Data were obtained from at least three repeats and presented as the mean ± SE in D, F, G, and I, ***P < .001, ****P < .0001. In all the panels, CK666 is an Arp2/3-specific inhibitor, while CK689 served as inactive analogue and DMSO as vehicle control

Article Snippet: Basic fibroblast growth factor (bFGF), rabbit anti- VE- cadherin phospho- specific [Tyr731] (NBP254766), and anti- human E- selectin (NBP1- 45545) Abs were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Transmigration Assay, Inhibition, Migration, Labeling, Control

Journal: The FASEB Journal

Article Title: E‐selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity

doi: 10.1096/fj.202000662rr

Figure Lengend Snippet: FIGURE 4 E-selectin manipulated Arp2/3 localization via associated cortactin. Immunoassay of phosphorylated Tyr466 and Tyr421 of cortactin (A) and their quantifications of the phosphorylation level (B) in 4 h LPS- and 30 minutes polymorphonuclear neutrophil (PMN)-treated scr or shE-sel human umbilical vein endothelial cell (HUVEC) monolayers. Data of at least three repeats were normalized to intact HUVECs and presented as the mean ± SE and compared using a t test. *P < .05. Typical images of in situ proximity ligation assay (PLA) for the paired E-selectin-cortactin (C), cortactin-Arp2/3 (D), and E-selectin-Arp2/3 (E) interplay in scr or shE-sel HUVEC monolayer. Three paired primary Abs were used separately: mouse anti-E-selectin and rabbit anticortactin, rabbit anticortactin and mouse anti-Arp3, or mouse anti-E-selectin and rabbit anti-Arp2 Abs. Two anti-Arp2/3 Abs raised from different species were used due as per requirement in PLA tests. Nuclei were stained with DAPI (blue). Quantification of dispersed red dots in C–E (F). Data were collected from at least three repeats from a total of 21–41 cells and presented as the mean ± SE. *P < .05

Article Snippet: Basic fibroblast growth factor (bFGF), rabbit anti- VE- cadherin phospho- specific [Tyr731] (NBP254766), and anti- human E- selectin (NBP1- 45545) Abs were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, In Situ, Proximity Ligation Assay, Staining

Journal: The FASEB Journal

Article Title: E‐selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity

doi: 10.1096/fj.202000662rr

Figure Lengend Snippet: FIGURE 6 Complementary roles of P-selectin for polymorphonuclear neutrophils (PMNs) during transendothelial migration (TEM). Effects of anti-P-selectin blocking antibody G1 on the TEM of PMNs across scr (A) or shE-sel (B) human umbilical vein endothelial cell (HUVEC) monolayer. Data were obtained from at least three repeats and presented as the mean ± SE (shadow bands). *P < .05. Time courses of TEM ratio in A and B were fitted by an exponential equation, y(t) = y∞{1 − exp(−t/τ)}. Estimated plateau value y∞ (C) and characteristic time τ (D) from individual fitting in each case, from at least three repeats, were lumped and presented as the mean ± SE. **P < .01

Article Snippet: Basic fibroblast growth factor (bFGF), rabbit anti- VE- cadherin phospho- specific [Tyr731] (NBP254766), and anti- human E- selectin (NBP1- 45545) Abs were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Blocking Assay

Journal: The FASEB Journal

Article Title: E‐selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity

doi: 10.1096/fj.202000662rr

Figure Lengend Snippet: FIGURE 7 Working model of E-selectin-dependent transendothelial migration (TEM) of polymorphonuclear neutrophils (PMNs). Negative regulation of E-selectins on PMN TEM is attributed to Arp2/3- and cortactin-mediated actin remodeling and junction repair. E-selectin knockdown weakens the activation of cortactin and Arp2/3, which prevents the formations of branched actin, lamellipodia protrusion, and reticular junction between endothelial cells (ECs). Subsequently, the adhesive function of vascular endothelial (VE)- cadherin is altered, enlarging the endothelial gap, and thus expedites the PMN transmigration

Article Snippet: Basic fibroblast growth factor (bFGF), rabbit anti- VE- cadherin phospho- specific [Tyr731] (NBP254766), and anti- human E- selectin (NBP1- 45545) Abs were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Knockdown, Activation Assay, Adhesive, Transmigration Assay