Fig. 6
Anantin effects do not depend on the activity of CaMKII, Cdk5 kinase or the phosphatase calcineurin. a Bar plot summarizes means of P2X3 current density values in control and after application of calcineurin inhibitor FK-506 (5 µM, 2.5 h), anantin (500 nM, 2 h) or their combination (n = 37, 36, 40, 31, respectively; *p < 0.05, Kruskal–Wallis test). Scheme on the right describes experimental protocol of anantin and FK-506 coapplication. FK-506 pretreatment does not prevent anantin-induced increase of P2X3 current density values. b Representative example of Western immunoblotting (right) and the histogram (left), summarizing P2X3 pSer relative optical density values in control, after application of anantin (500 nM, 2 h), FK-506 (5 µM, 2.5 h), or their combination (n = 3; *p < 0.05, Kruskal–Wallis test). c Histograms show mean current density values of P2X3 receptors in control, after 24 h Cdk5 silencing with small interfering RNAs, after anantin application (500 nM, 3 h), or after a combination of those two treatments (n = 32, 31, 40, 30, respectively; *p < 0.05, Kruskal–Wallis test). Scheme on the right describes experimental protocol of combined siCdk5 and anantin treatments. Cdk5 RNA silencing does not prevent anantin from upregulating P2X3 receptor activity. d Representative example of Western immunoblotting (right) and histogram (left), summarizing P2X3 pSer (relative optical density values) in control, after application of anantin, 24 h siCdk5, or a combination of these treatments (n = 3; *p < 0.05, Kruskal–Wallis test); β-tubulin used as loading control of the total extract. e Histograms compare mean current density values of P2X3 receptors in control and after application of CaMKII inhibitor KN-93 (5 µM, 3.5 h), anantin (500 nM, 2 h), or their combination (n = 29, 26, 40, 28, respectively; *p < 0.05, Kruskal–Wallis test). Scheme on the right describes experimental protocol of anantin and KN-93 coapplication. KN-93 does not influence anantin-mediated increase in P2X3 current density. f Representative example of Western immunoblotting (right) and histograms (left), summarizing P2X3 pSer (relative optical density values) in control and after application of anantin (500 nM, 2 h), KN-93 (5 µM, 3.5 h), or KN-93 together with anantin (n = 3; *p < 0.05, Kruskal–Wallis test)