Human HB-EGF Antibody

Detection of HB-EGF by Flow Cytometry CXCL12 modifies HB-EGF expression in mononuclear phagocytes. Human mononuclear phagocytes (Mø) were cultured alone or in the presence of 200 ng/mL CXCL12. Cells were collected after 20 minutes, 2 hours and 24 hours; cell-free supernatants were collected after 24 hours and the levels of soluble HB-EGF protein were measured using a specific ELISA. (A) Flow cytometric analysis showing that CXCL12-stimulated Mø released HB-EGF (after 20 minutes) and up-regulated its expression (after 24 hours). (B) Northern blot analysis on Mø and neutrophils (PMN, used as negative control) collected after 2 hours of stimulation with CXCL12. Only Mø produced detectable levels of HB-EGF mRNA in basal conditions, and HB-EGF transcripts were up-regulated upon stimulation. After 24 hours, the mRNA up-regulation persisted (data not shown). (C) CXCL12 treatment induced Mø to release HB-EGF into the culture medium (p < 0.05). Representative pictures or the means ± SD out of 10 experiments are shown. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/20946648), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of HB-EGF by Western Blot CXCL12 modifies HB-EGF expression in mononuclear phagocytes. Human mononuclear phagocytes (Mø) were cultured alone or in the presence of 200 ng/mL CXCL12. Cells were collected after 20 minutes, 2 hours and 24 hours; cell-free supernatants were collected after 24 hours and the levels of soluble HB-EGF protein were measured using a specific ELISA. (A) Flow cytometric analysis showing that CXCL12-stimulated Mø released HB-EGF (after 20 minutes) and up-regulated its expression (after 24 hours). (B) Northern blot analysis on Mø and neutrophils (PMN, used as negative control) collected after 2 hours of stimulation with CXCL12. Only Mø produced detectable levels of HB-EGF mRNA in basal conditions, and HB-EGF transcripts were up-regulated upon stimulation. After 24 hours, the mRNA up-regulation persisted (data not shown). (C) CXCL12 treatment induced Mø to release HB-EGF into the culture medium (p < 0.05). Representative pictures or the means ± SD out of 10 experiments are shown. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/20946648), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of HB-EGF by Flow Cytometry Knockdown of GM-CSF protein levels after siRNA application in cancer cells. HeLa/DLD-1 cells were transfected with control siRNA (1/1*, 2/2*) or GM-CSF siRNA (3/3*, 4/4*) and cultured in the absence or presence of 25 ng/mL HB-EGF. The numbers indicate the culture conditions and the corresponding supernatants (SN) used for ELISA or cell stimulation. (A) Blockade of GM-CSF production in cultures of HeLa/DLD-1 cells transfected with GM-CSF siRNA was confirmed by immunocytochemistry (2/2* vs. 4/4*) and ELISA (left side; 2/2* vs. 4/4*, p < 0.05). (B) SN from GM-CSF-silenced HeLa/DLD-1 did not induce HB-EGF expression in mononuclear phagocytes (Mø), as revealed by flow cytometry (2/2* vs. 4/4*) and ELISA (left side; 2/2* vs. 4/4*, p < 0.05). (C) Mø stimulated with SN from GM-CSF-silenced HeLa/DLD-1 cells released SN less effective at inducing GM-CSF in non-silenced cancer cells, as determined by ELISA (see Methods section; SN2 vs. SN4, p < 0.05). Representative pictures or the means ± SD out of 5 experiments are shown. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/20946648), licensed under a CC-BY license. Not internally tested by R&D Systems.