Figure 3

The effects of BAY-747 and runcaciguat on GluA1-containing AMPAR mobilization to the surface in an ex-vivo acquisition-like cLTP model. (A) Incubation with 100 nM BAY-747 in combination with WS enhanced GluA1-containing AMPAR mobilization as measured by the surface GluA1/total GluA1 ratio, compared to vehicle, 100 nM BAY-747 only, and WS only. (B) The phosphorylation levels of S845 on GluA1 were enhanced by WS and a combination of BAY-747 and WS, yet BAY-747 could not further enhance S845 phosphorylation in combination with WS when compared to WS only. (C) Representative image of a full BAY-747 western blot. (D) Incubation with 10 nM runcaciguat in combination with WS partially enhanced GluA1-containing AMPAR mobilization to the surface as measured by the surface GluA1/total GluA1 ratio, since the mobilization measure was significantly higher than vehicle and runcaciguat only, but not to WS only while WS in itself did not affect AMPAR mobilization. (E) The phosphorylation levels of S845 on GluA1 were enhanced by the combination of runcaciguat and WS when compared to WS only, vehicle, and runcaciguat only. (F) representative image of a full western blot. Each western blot contained an identical loading control with a known protein concentration as an internal correction for signal intensity/background differences between blots. Since GAPDH barely associates with membrane proteins, the membrane protein fraction contained no or very little GAPDH on the western blot. Only pS845 of the total protein samples was analyzed. BAY-747 was incubated at a concentration of 100 nM, while runcaciguat was incubated at a concentration of 10 nM. Data are represented as mean + SEM. n = 15–23. *P < 0.05; **P < 0.01; ***P < 0.001 as measured with post-hoc LSD tests. For scans of all western blots analyzed, please see Supplemental WB Figs. A1–A3 for BAY-747 and Supplemental WB Figs. B1–B5 for Runcaciguat.