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. Author manuscript; available in PMC: 2014 Apr 15.

Published in final edited form as: Biochem Pharmacol. 2013 Jan 31;85(8):1171–1181. doi: 10.1016/j.bcp.2013.01.021

Kinetics of MRS5218 binding to the hA₃AR expressed in CHO cells determined using FCM. Each graph represents the average of three experiments. A) Association in the absence or presence of 0.4 M sucrose at 37°C and in absence of sucrose at 4°C. Cells were incubated with 30 nM MRS5218 for different time intervals from 5 min to 180 min. The observed association rate constant (K) was 0.039 min⁻¹ for the incubation in absence of sucrose at 37°C, K = 0.026 min⁻¹ for the incubation in presence of sucrose at 37°C and K = 0.013 min⁻¹ for the incubation at 4°C, determined by fitting a one phase exponential association equation to the obtained data. B) Association followed by dissociation of MRS5218 (30 nM) initiated at 60 min by the addition of 10 µM MRS1220, measured in the presence of 0.4 M sucrose at 37°C. The observed t_1/2 of the dissociation was 21.4 min. C) Internalization kinetics of 30 nM MRS5218. Cells were incubated with 30 nM MRS5218 for different time intervals from 5 min to 90 min. Internalized amount (%_int) was calculated as the acid insensitive fluorescence at x time point (MESF_x) compared to total MESF (MESF_total) and corrected with the nonspecific binding (MESF_nonspec): %_int = (MESF_x - MESF_nonspec) / (MESF_total - MESF_nonspec). The acid-insensitive binding of MRS5218 was measured after removing cell-surface bound ligand by 3 × 5 min acid wash (pH 3.5). After 90 min of incubation, the percentage of internalized MRS5218 was 47.6%. The t_1/2 of the internalization was 12.9 min.

Kinetics of MRS5218 binding to the hA₃AR expressed in CHO cells determined using FCM. Each graph represents the average of three experiments. A) Association in the absence or presence of 0.4 M sucrose at 37°C and in absence of sucrose at 4°C. Cells were incubated with 30 nM MRS5218 for different time intervals from 5 min to 180 min. The observed association rate constant (K) was 0.039 min⁻¹ for the incubation in absence of sucrose at 37°C, K = 0.026 min⁻¹ for the incubation in presence of sucrose at 37°C and K = 0.013 min⁻¹ for the incubation at 4°C, determined by fitting a one phase exponential association equation to the obtained data. B) Association followed by dissociation of MRS5218 (30 nM) initiated at 60 min by the addition of 10 µM MRS1220, measured in the presence of 0.4 M sucrose at 37°C. The observed t_1/2 of the dissociation was 21.4 min. C) Internalization kinetics of 30 nM MRS5218. Cells were incubated with 30 nM MRS5218 for different time intervals from 5 min to 90 min. Internalized amount (%_int) was calculated as the acid insensitive fluorescence at x time point (MESF_x) compared to total MESF (MESF_total) and corrected with the nonspecific binding (MESF_nonspec): %_int = (MESF_x - MESF_nonspec) / (MESF_total - MESF_nonspec). The acid-insensitive binding of MRS5218 was measured after removing cell-surface bound ligand by 3 × 5 min acid wash (pH 3.5). After 90 min of incubation, the percentage of internalized MRS5218 was 47.6%. The t_1/2 of the internalization was 12.9 min.