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To further detect the binding site of paeoniflorin with HSA, the competitive binding experiment was carried out.
Down regulation of mAChR in stably transfected HEK cells was measured using the same protocol except that 70 80% confluent 15 cm plates were split into two 6 well plates and the binding experiment was carried out the following day.
Cell surface expression in stably transfected HEK cells was measured with the same protocol except that 70 80% confluent 15 cm plates were split into two 6 well plates and the binding experiment was carried out the following day.
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Binding experiments were carried out with a Monolith NT.115 Series instrument (Nano Temper Technologies GMBH).
Serum samples were diluted, polyclonal IgG was captured, and binding experiments were carried out as previously described [31], [32].
Competitive binding experiments were carried out with BSA-warfarin and ibuprofen complexes in presence of SI and SA forms and the results are presented in Figure 5A & 5B.
All saturation binding experiments were carried out at 25°C in 0.5 mL Tris buffer (50 mM, pH 7.4) containing 10 15 µg of protein.
Similarly, actin binding experiments were carried out for inactive and active full-length vinculin (15 µM), metavinculin (14 µM), or MV-ΔH1 (15 µM) with F-actin (30 µM).
All saturation binding experiments were carried out at 25°C in 0.5 mL Tris buffer (50 mM, pH 7.4) containing 50 70 µg protein.
Binding experiments were carried out at 4°C for 1 hr.
All binding experiments were carried out at 30°C with 1,000 rpm stirring.
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