Characterization of a panel of six β2-adrenergic receptor antibodies by indirect immunofluorescence microscopy

<p>Abstract</p> <p>Background</p> <p>The β₂-adrenergic receptor (β₂AR) is a primary target for medications used to treat asthma. Due to the low abundance of β₂AR, very few studies have reported its localization in tissues. However, the intracellular location of β₂AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β₂AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β₂AR in primary cell cultures.</p> <p>Methods</p> <p>A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β₂AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β₂AR were identified and used to localize native β₂AR in primary cultures of rat airway smooth muscle and epithelial cells. β₂AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol ^([3H]DHA).</p> <p>Results</p> <p>Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β₂AR (Ab-Bethyl) specifically recognized human but not rat β₂AR. An antibody developed against the C-terminal domain of the mouse β₂AR (Ab-sc570) specifically recognized rat but not human β₂AR. An antibody developed against 78 amino acids of the C-terminus of the human β₂AR (Ab-13989) was capable of recognizing both rat and human β₂ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β₂AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface.</p> <p>Conclusion</p> <p>Antibodies have been identified that recognize human β₂AR, rat β₂AR or both rat and human β₂AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β₂AR in tissues.</p

Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

Background: The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. Methodology/Principal Findings: In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: ‘‘rigidity dependent’ ’ (those which show an increase in cell growth as extracellular rigidity is increased), and ‘‘rigidity independent’’ (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. Conclusions/Significance: These observations suggest that the mechanical properties of the matrix environment play

Characterization of a panel of six β-adrenergic receptor antibodies by indirect immunofluorescence microscopy-4

either untreated (A, C) or treated (B, D) with isoproterenol for 4.5 h in parallel, fixed and processed for microscopy (Axioskop 2 plus epifluorescent microscope).<p><b>Copyright information:</b></p><p>Taken from "Characterization of a panel of six β-adrenergic receptor antibodies by indirect immunofluorescence microscopy"</p><p>http://respiratory-research.com/content/9/1/32</p><p>Respiratory Research 2008;9(1):32-32.</p><p>Published online 18 Apr 2008</p><p>PMCID:PMC2383888.</p><p></p

Characterization of a panel of six β-adrenergic receptor antibodies by indirect immunofluorescence microscopy-1

either untreated (A, C) or treated (B, D) with isoproterenol for 4.5 h in parallel, fixed and processed for microscopy (Axioskop 2 plus epifluorescent microscope). (E) Sequence comparison of the last 15 amino acids of the human, rat and mouse βAR.<p><b>Copyright information:</b></p><p>Taken from "Characterization of a panel of six β-adrenergic receptor antibodies by indirect immunofluorescence microscopy"</p><p>http://respiratory-research.com/content/9/1/32</p><p>Respiratory Research 2008;9(1):32-32.</p><p>Published online 18 Apr 2008</p><p>PMCID:PMC2383888.</p><p></p

Characterization of a panel of six β-adrenergic receptor antibodies by indirect immunofluorescence microscopy-2

either untreated (A, C) or treated (B, D) with isoproterenol for 4.5 h in parallel, fixed and processed for microscopy using a LSM510 confocal microscope. HEK 293 cell lines expressing different levels of human βAR were processed and analyzed by wide field (E) and confocal (F) microscopy to establish the range over which receptor number and fluorescence signal intensity was linear.<p><b>Copyright information:</b></p><p>Taken from "Characterization of a panel of six β-adrenergic receptor antibodies by indirect immunofluorescence microscopy"</p><p>http://respiratory-research.com/content/9/1/32</p><p>Respiratory Research 2008;9(1):32-32.</p><p>Published online 18 Apr 2008</p><p>PMCID:PMC2383888.</p><p></p

Characterization of a panel of six β-adrenergic receptor antibodies by indirect immunofluorescence microscopy-3

βAR (Ab-13989) (B) and anti-smooth muscle α-actin antibodies (A). C – merged image. Airway epithelial cells were harvested from rat lungs, fixed and double-labeled with βAR (Ab-13989, E and Ab-sc570, H) and E-cadherin antibodies (D, G, and J). Ab-13989 and Ab-sc570 demonstrated a similar pattern of staining in primary cultures (E and H). Preincubation of Ab-sc570 with neutralizing peptide (sc570p) abrogated the staining (K). Panels F, I, L are merged images.<p><b>Copyright information:</b></p><p>Taken from "Characterization of a panel of six β-adrenergic receptor antibodies by indirect immunofluorescence microscopy"</p><p>http://respiratory-research.com/content/9/1/32</p><p>Respiratory Research 2008;9(1):32-32.</p><p>Published online 18 Apr 2008</p><p>PMCID:PMC2383888.</p><p></p