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Figure 2 Inhibition of receptor-localized phosphoinositide 3-kinase (PI3K) activity does not affect downstream signaling pathways. (a) Northern blotting analysis showing ß-adrenergic receptor (ß₁AR) levels in 12-week-old wild type (WT), calsequestrin (CSQ), and CSQ/catalytically inactive PI3K (CSQ/PI3K_inact) hearts (upper panel); equal loading of the different RNA samples was confirmed by methylene blue staining of nylon membranes (bottom panel). (b to e) Mitogen-activated protein kinase activation was determined in WT, CSQ, and CSQ/PI3K_inact hearts by the ability to in vitro phosphorylate myelin binding protein (MBP) or recombinant glutathione S transferase-proto-oncogene c-Jun (GST-cJun). Representative kinase assays and relative densitometric evaluation of at least six independent experiments are shown for extracellular signal-related kinase (ERK) (b), c-Jun N-terminal kinase (JNK) (c), p38 (d), and p38ß (e). Western blotting was carried out to evaluate total protein levels of each kinase (b to d). *p < 0.01 for CSQ or CSQ/PI3K_inact versus WT (analysis of variance with Neuman-Keuls correction). (f) Western blotting analysis showing similar activation of protein kinase B (PKB) and glycogen synthase kinase (GSK) under basal conditions in single CSQ and binary CSQ/PI3K_inact mice. IB = immunoblotting; IP = immunoprecipitation.

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