The association of FKBP12.6 is reduced upon in vitro PKA phosphorylation and incubation with rapamycin. (A) Representative co-IP blots of RyR2s isolated from sheep heart that were subjected to in vitro PKA phosphorylation either before (PKA-PP1) or after (PP1-PKA) dephosphorylation by PP1 or RyR2 subjected to rapamycin (Rap; see Materials and methods). As controls we show an untreated sample (neither dephosphorylated nor subsequently phosphorylated, untreated) and buffer incubation controls (PP1-PKA ctrl and PKA-PP1 ctrl). Blots were probed with antibodies to RyR2 and to an antibody that detects FKBP12.6 and 12.0. (B) Corresponding average data for FKBP12.0 and FKBP12.6 dissociation from RyR2. Band densities were normalized to RyR2 in each lane and expressed relative to normalized band densities of untreated RyR2. Data in bar graphs are shown as mean ± SEM from five independent experiments. P values denote a significant difference in FKBP association with RyR2 compared with untreated RyR2, which are, for FKBP12 (left to right), 0.75, 0.70, 0.57, 0.54, and 0.40, and for FKBP12.6, 0.39, 0.61, 005, 0.14, and 0.00008. Source data are available for this figure: SourceData F3.