mouse anti β2ar antibody  (Santa Cruz Biotechnology)

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Agonists and hydrogen peroxide mediate hyperoxidation of β2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to β2-agonists in constrictive airway disorders.

doi: 10.1016/j.biopha.2023.115763

Figure Lengend Snippet: Fig. 1. β2AR agonism stimulates H2O2 production in human airway epithelial cells in a NOX dependent manner. Agonism of β2AR with increasing ISO concentrations initiates concentration-dependent increases in real-time H2O2 production. (A) ISO (10 µM) significantly increases H2O2 production (p < 0.001 via One-way ANOVA with Tukey’s post-hoc) versus vehicle-control in CALU-3, A549, SAEC and A-SAEC, as detected by the extracellular H2O2-selective probe Amplex Red (n = 3). H2O2 (0.1 µM) was used as an internal positive control to ensure probe viability (data not shown). Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test and * ** denotes p < 0.001 versus the vehicle-control condition. (B-D) ISO (1 µM) significantly increased (p < 0.001) H2O2 production in CALU3 (B), SAEC (C) and A-SAEC (D) cells and pretreatment with the selective NOX inhibitor, VAS2870 (5 µM; 30 min) significantly attenuated real- time H2O2 generation in CALU-3 (p < 0.05 via ANOVA with Tukey’s post-hoc) and SAEC (p < 0.001 via ANOVA with Tukey’s post-hoc) (n = 3). The ISO-induced real-time H2O2 generation remained unaltered in the presence of VAS2870 in A-SAEC (n = 3). Compared to the ISO-alone condition, AUC analysis reveals a 30.7% and 148% reduction in ISO-induced real-time H2O2 generation in the presence of VAS2870 in CALU-3 (B, lower), and SAEC (C, lower), respectively (n = 3). Values represent average of three independent experiments (n = 3), performed in triplicates, vehicle control (unstimulated) values were subtracted from each data point for baseline correction. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test and * * denotes p < 0.01, and * ** denotes p < 0.001 versus the vehicle-control condition, while # denotes p < 0.05, ### denotes p < 0.001, and #### denotes p < 0.0001 versus the respective ISO-treated condition. (E) ISO (10 µM) significantly increases H2O2 production versus vehicle-control in CALU-3, SAEC and A-SAEC, as detected by the intracellular H2O2-selective probe AbGreen. Cells were pretreated with VAS2870 (VAS; 5 µM) or the β2AR inverse agonist ICI-118,551 (ICI; 10 µM) for 30 min, both of which significantly inhibit fluorescent intensity, demonstrating β2AR- and NOX-dependent H2O2 generation. H2O2 (0.1 µM) served as positive control in each experiment. Graphs represent quantified fluorescent intensity from 4X images from three independent experiments (n = 3) performed in triplicates. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test and * denotes p < 0.05, and * * denotes p < 0.01 versus the vehicle-control condition, while # denotes p < 0.05, and ## denotes p < 0.01 versus the respective ISO-treated condition, as shown.

Article Snippet: Prepared lysates were tumbled with 10 μl (0.2 μg/μl) anti-human β2AR mouse monoclonal antibody (E3, Santa Cruz Biotechnology) for 2 h at 4 ◦C and 20 μl of resuspended protein A/G -agarose beads (Thermo Fischer Scientific) were added and tumbled overnight at 4 ◦C.

Techniques: Concentration Assay, Control, Positive Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Agonists and hydrogen peroxide mediate hyperoxidation of β2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to β2-agonists in constrictive airway disorders.

doi: 10.1016/j.biopha.2023.115763

Figure Lengend Snippet: Fig. 2. Exogenous H2O2 and β-receptor agonism induces β2AR Cys-S-sulfination in human SAEC. (A) The ability of acute agonism of β2AR to induce cysteine-S- sulfination was assessed via the clickable S-sulfinic acid-selective probe DiaAlk. SAEC or A-SAEC (4 ×105 cells/well) were seeded in 6-well plates and agonized with (A) ISO (10 μM) for 5–60 min (n = 3) or (B) salbutamol (albuterol; SAL) (10 μM) for 15 or 60 min (n = 3). β2AR cysteine-S-sulfination was assessed by β2AR immunoprecipitation and selective labeling with 1 mM DiaAlk, followed by conjugated with biotin utilizing click chemistry, and detection via streptavidin-HRP, as discussed in the materials and methods. Both ISO (A) and SAL (B) significantly increased β2AR hyperoxidation to cysteine-S-sulfinic acids (upper panels). The amount of β2AR (middle panels) within each reaction is shown as an input control and β-actin (lower panels) is utilized to denote equal protein input and loading. (C) The effects of chronic ISO agonism or H2O2 were also assessed and cells were treated every 12 h with for seven days with either ISO (10 µM) or H2O2 (10 µM). The media was replenished every 2–3 days to prevent nutrient depletion-mediated starvation. Both SAEC and A-SAEC exhibit significantly elevated DiaAlk-biotin labeled β2AR (upper panels), indicative of cysteine-S-sulfination, following the seven-day treatment paradigm with both ISO and H2O2. The amount of β2AR (middle panels) within each reaction is shown as an input control and β-actin (lower panels) is utilized to denote equal protein input and loading.

Article Snippet: Prepared lysates were tumbled with 10 μl (0.2 μg/μl) anti-human β2AR mouse monoclonal antibody (E3, Santa Cruz Biotechnology) for 2 h at 4 ◦C and 20 μl of resuspended protein A/G -agarose beads (Thermo Fischer Scientific) were added and tumbled overnight at 4 ◦C.

Techniques: Immunoprecipitation, Labeling, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Agonists and hydrogen peroxide mediate hyperoxidation of β2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to β2-agonists in constrictive airway disorders.

doi: 10.1016/j.biopha.2023.115763

Figure Lengend Snippet: Fig. 3. Low concentrations of exogenous H2O2 enhance, while 1 mM H2O2 inhibits ISO-induced cAMP production. (A) Real-time cAMP was assessed in the presence of various H2O2 concentrations introduced 10 min (arrows) following agonism with ISO. While H2O2 concentrations spanning 0.1–1 µM enhanced cAMP production, higher concentrations decreased it, and the 1 mM H2O2 concentration significantly inhibited ISO-induced cAMP production in both (A) SAEC (p < 0.001) and A-SAEC (p < 0.001). Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test and ### denotes p < 0.001 versus the respective ISO- treated condition (n = 3). (B) To ensure that the effect of 1 mM H2O2 was not due to induction of cell death, cell viability was assessed 4 h following agonism with ISO via trypan blue exclusion and there was no significant effect of the 1 mM H2O2 treatment on cell viability in either SAEC or A-SAEC (pooled data from n = 3). (C) The effects of 1 mM H2O2 were also assessed on variable ISO concentrations (0.1 μM - 100 μM) and in both SAEC and A-SAEC, 1 mM H2O2 added 10 min (arrows) following agonism with ISO abolished the cAMP-generating effects of all concentrations of ISO (p < 0.0001 for both cell types, one-way ANOVA, n = 3), and this effect remained for the 4 h observation duration (not shown). (D) To ensure that the effect of 1 mM H2O2 was not due to induction of cell death, cell viability was assessed 4 h following agonism with ISO via trypan blue exclusion and there was no significant effect of the 1 mM H2O2 treatment on cell viability in either SAEC or A-SAEC (pooled data from n = 3). (E) Given that these results reflect effects of H2O2 treatment following agonism with ISO, we also assessed the outcomes of ISO- induced cAMP formation in cells pre-incubated with H2O2 (1 mM) for 1 min prior to agonism with ISO, as we previously showed this was enough to induce cysteine- S-sulfenation of β2AR [18,28]. In this case, H2O2 also significantly inhibited cAMP formation in both SAEC (p < 0.0001) and A-SAEC (p < 0.0001), with no sig nificant effect on cell viability (F) (n = 3).

Article Snippet: Prepared lysates were tumbled with 10 μl (0.2 μg/μl) anti-human β2AR mouse monoclonal antibody (E3, Santa Cruz Biotechnology) for 2 h at 4 ◦C and 20 μl of resuspended protein A/G -agarose beads (Thermo Fischer Scientific) were added and tumbled overnight at 4 ◦C.

Techniques: Concentration Assay, Incubation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Agonists and hydrogen peroxide mediate hyperoxidation of β2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to β2-agonists in constrictive airway disorders.

doi: 10.1016/j.biopha.2023.115763

Figure Lengend Snippet: Fig. 5. Altered real-time β2AR-mediated cAMP formation in SAECs. (A-B) Asthma-derived SAEC exhibit significantly higher cAMP formation (p < 0.001) compared to healthy SAEC upon stimulation with (A) forskolin (FSK) or (B) ISO. Each point in the graph represents mean of 3 independent experiments performed in triplicates (n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test and significance denoted as * * p < 0.01 or * ** p < 0.001 versus healthy SAEC. Given the variable concentrations of cAMP in each cell type, data are normalized to a percentage of the unstimulated SAEC response ± SD. (C, upper) Over a 4 h time period, both basal and ISO-induced (1 µM) real-time cAMP formation were significantly elevated in A-SAEC compared to healthy SAEC (p < 0.001, two-way ANOVA). (C, lower) AUC analysis over the entire time-frame also revealed significantly higher real-time cAMP formation in A-SAEC vs SAEC (p < 0.001, two-way ANOVA). (D-E) Agonism of β2AR with ISO (1 µM) induces significant increases in real-time cAMP formation in (D) SAEC and (E) A-SAEC, and this effect was abolished (p < 0.0001) in the presence of β2AR inverse agonist, ICI-118,551 (100 μM) introduced 10 min (arrows) following agonism with ISO, indicating that the effects seen are β2AR specific. Notably, basal cAMP formation in the absence of agonist was also significantly reduced by ICI-118,551 treatment, demonstrating a significant contribution of β2AR to constitutive cAMP signaling in both SAEC and A-SAEC. (D-E, lower) AUC analysis over the entire time-frame also revealed the effects of ICI-118,551 alone and with ISO in A-SAEC vs SAEC. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test and significance denoted as * ** p < 0.001, as shown. (F-G) ICI-118,551 treatment 10 min (black arrows) following agonism with ISO blocks both basal and ISO effects at various concentrations of ISO (0.1–100 µM) in both (F) SAEC and (G) A-SAEC. To ensure that cells were viable after 4 h, they were re-challenged with 10 µM FSK/ 100 µM IBMX (red arrows), and responded with increases in real-time cAMP generation. (H) To confirm that the effects of ICI-118,551 were not due to induction of cell death, cell viability was assessed 4 h following agonism with ISO via trypan blue exclusion and there was no significant effect of ISO-118,551 treatment on cell viability in either SAEC or A-SAEC (pooled data from n = 3).

Article Snippet: Prepared lysates were tumbled with 10 μl (0.2 μg/μl) anti-human β2AR mouse monoclonal antibody (E3, Santa Cruz Biotechnology) for 2 h at 4 ◦C and 20 μl of resuspended protein A/G -agarose beads (Thermo Fischer Scientific) were added and tumbled overnight at 4 ◦C.

Techniques: Derivative Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Agonists and hydrogen peroxide mediate hyperoxidation of β2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to β2-agonists in constrictive airway disorders.

doi: 10.1016/j.biopha.2023.115763

Figure Lengend Snippet: Fig. 6. A-SAEC exhibit lower adenylyl cyclase V/VI and enhanced PDE4 expression, but similar β2AR, Gαs, and Gαi, SOD1-3, and catalase expression. Immunoblot analysis of SAEC and A-SAEC protein lysates for detection of proteins that could contribute to alterations in cAMP formation. Expression of (A) β2AR (56 – 85 kDa represents native and variably glycosylated β2AR species) and (B) Gαs (45 and 52 kDa bands represent short and long isoforms, respectively) were unaltered in SAEC compared to A-SAEC (n = 3, each). (C) Expression of ACV/VI was slightly decreased in A-SAEC compared to SAEC (n = 4), while expression of Gαi1/2 isoform (n = 3) was not significantly different (D). (E) Expression of PDE4 was significantly heightened in A-SAEC compared to SAEC (n = 4). β-actin served as a loading control for each representative immunoblot. (F) Expression of SOD1, SOD2, SOD3, and was not significantly altered, while catalase expression was visually slightly, though not significantly, higher in A-SAEC. SOD2–3 were probed from the same blots, hence, actins are the same.

Article Snippet: Prepared lysates were tumbled with 10 μl (0.2 μg/μl) anti-human β2AR mouse monoclonal antibody (E3, Santa Cruz Biotechnology) for 2 h at 4 ◦C and 20 μl of resuspended protein A/G -agarose beads (Thermo Fischer Scientific) were added and tumbled overnight at 4 ◦C.

Techniques: Expressing, Western Blot, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Agonists and hydrogen peroxide mediate hyperoxidation of β2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to β2-agonists in constrictive airway disorders.

doi: 10.1016/j.biopha.2023.115763

Figure Lengend Snippet: Fig. 7. Elevated PDE expression in asthma-SAEC modulates alterations in basal and β2AR-induced cAMP formation. In the absence of IBMX (100 μM) basal cAMP response in (A) A-SAEC was significantly attenuated (p < 0.001) in comparison to healthy SAEC, illustrating the influence of uninhibited and elevated PDE4 activity in decreasing the cAMP signal. The basal cAMP response was abolished in the presence of β2AR inverse agonist ICI-118,551 in both SAEC (p < 0.05) and A- SAEC (p < 0.05). In addition, ISO-induced cAMP formation in SAEC and A-SAEC, with and without IBMX, respectively was completely diminished by ICI-118,551 (100 μM, 5 min) preincubation. Statistical analysis was performed using ANOVA with Tukey’s post-hoc test, and significance is denoted as * p < 0.05 or * ** p < 0.001, as shown, n = 3, with each performed in triplicate. Given the variable concentrations of cAMP in each cell type, data are normalized to a percentage of the SAEC response ± SD. (B) The concentration-responses demonstrate decreased cAMP formation in A-SAEC compared to SAEC, with ICI-118,551 flattening the cAMP responses to ISO in both SAEC (p < 0.001) and A-SAEC (p < 0.001). Statistical analysis was performed using ANOVA with Tukey’s post-hoc test, and sig nificance is denoted as * p < 0.05 compared to the SAEC + IBMX condition and ### p < 0.001 compared to the respective condition lacking ICI-118,551. Each experiment was independently performed in triplicate twice, and given the variable concentrations of cAMP in each cell type, data are normalized to a percentage of the SAEC response ± SD.

Article Snippet: Prepared lysates were tumbled with 10 μl (0.2 μg/μl) anti-human β2AR mouse monoclonal antibody (E3, Santa Cruz Biotechnology) for 2 h at 4 ◦C and 20 μl of resuspended protein A/G -agarose beads (Thermo Fischer Scientific) were added and tumbled overnight at 4 ◦C.

Techniques: Expressing, Comparison, Activity Assay, Concentration Assay