Fig. 3: PAGln effect on binding of orthosteric ligand [³H]-propranolol and ISO-induced dynamic mass redistribution (DMR) on β2AR.

A Competition binding dose-response curve of PAGln alone (Black), isoproterenol (ISO) alone (Blue), or ISO with 100 µM PAGln (Red) to the β2-HEK293 cell membranes with [³H]-propranolol (P < 0.0001; EC₅₀ Blue vs Red). PAGln pre-incubation in β2-HEK293 displays 2.4 ± 0.07-fold increase (P = 0.001) in the EC₅₀ of the dose-response as compared to ISO alone control. Two-tailed unpaired Welch’s student t-test was used to obtain the fold change P-value when comparing the EC₅₀ values for ISO and ISO+PAGIn from three different experiments (ISO vs ISO+PAGIn; P = 0.001). B Saturation-binding in β2-HEK293 cell membranes with increasing concentrations of orthosteric ligand [³H]-propranolol alone (Blue), [³H]-propranolol along with 100 µM PAGln (Red) or 10 mM ISO (Purple) (P = 0.002; K_d Blue vs Re_d). PAGln pre-incubation in β2-HEK293 displays 2.1 ± 0.3-fold decrease (P = 0.01) in the K_d as compared to ISO alone control. Two-tailed unpaired Welch’s student t-test was used to obtain the fold change P-value when comparing the K_d values for [³H]-propranolol and [³H]-propranolol +PAGIn from three different experiments ([³H]-propranolol vs [³H]-propranolol +PAGIn; P = 0.01). C ISO-induced dynamic mass redistribution (DMR) dose-response in β2-HEK293 cells alone (Blue) and in presence of 100 µM PAGln (Red). DMR response of 0.1 µM ISO and 0.1 µM ISO along with 100 µM PAGln are highlighted in the dashed box right side. EC₅₀ of ISO and ISO+PAGln are indicated (P = 0.98; EC₅₀ Blue vs Red). Data points represent the mean ± SEM (for PAGln alone; Fig. 3A) and mean ± SD (for the rest) of the indicated number (n) of experiments. P values (top left) for EC₅₀ comparisons (Blue vs Red) as well as the standard error of the mean EC₅₀ (SEM) were determined with a non-linear regression method and the significance of the fold changes was analyzed using two-tailed unpaired Welch’s student t-test. Nonparametric two-tailed Mann–Whitney test was used for non-pairwise comparisons (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). All reported P values are two-sided. A P-value of <0.05 was considered significant in this study.