Phosphorylation and nuclear signalling of RSKs in cardiomyocytes
(A) Cardiomyocytes were exposed to ET-1 or A61603 for the times indicated. Total extracts were immunoblotted with antibodies against total RSKs or RSKs phosphorylated on Thr573, Thr359/Ser363 or Ser380. Blots are representative of at least three experiments with independent myocyte preparations. (B) Densitometric analysis of the blots in (A). Data were normalized to the maximum values. Results are means±S.E.M. for three (ET-1) or five (A61603) experiments with different cardiomyocyte preparations. (C and E) Cardiomyocytes were unstimulated (Con), or exposed to ET-1, PE or A61603 for the times indicated (C) or for 5 min (E). Cysotolic and nuclear proteins were immunoblotted with antibodies against phospho-RSK(Thr573) (P-RSKs) or total RSKs (T-RSKs). Blots are representative of at least three experiments with different cardiomyocyte preparations. (D) Densitometric analysis of the blots in (C). The graphs on the left-hand side show data normalized to the maximum values. The graphs on the right-hand side show the relative percentage in each fraction. Results are means±S.E.M. for three experiments with different cardiomyocyte preparations. *P<0.05, #P<0.01 and **P<0.001 relative to zero time. (F) Immunoblots of nuclear proteins following stimulation with ET-1, A61603 or PE (5 min) using goat polyclonal antibodies against RSK1 (sc-231) or RSK2 (sc-1430), or antibodies against phospho-ERK1/2 (P-ERKs) or total ERK1/2 (T-ERKs). The experiment was repeated twice with different cardiomyocyte preparations with similar results. (G) Immunoblots of nuclear proteins following stimulation with ET-1 or A61603 using rabbit polyclonal antibodies against RSK1 (#9333) or RSK2 (#9340) or antibodies against GAPDH or CREB. The experiment was repeated twice with different cardiomyocyte preparations with similar results. (H) Densitometric analysis of the data from (F) and (G). Results are means±S.E.M. for at least three observations with different cardiomyocyte preparations. *P<0.01 and **P<0.001 relative to zero time; #P<0.01 relative to A61603 at the same time.