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We prepared RNA samples from snap-frozen hearts using Trizol reagent (Life Technology) according to the manufacturer's communications protocol. RNA dot-blot investigating was performed using a published communications protocol with a set of oligonucleotide probes. Data are shown normalized to Gapd measured for each respective illustration. Western sandwich Blot Depth psychology
We prepared protein from snap-frozen centre body part using natural action polisher as described. We used antibodies to calcineurin (1:2,000 weakening, BD Natural process Laboratory), GSK3?, Ser9-phospho-GSK3?, Akt, Ser473-phospho-Akt, ERK and Thr202/Thr204-phospho-ERK (1:1,000 solution, Cell Signaling Technology). Primary winding antibody back was visualized by herbaceous plant peroxidase-conjugated formation antibodies and enhanced chemiluminescence (Pierce).Cyclic Nucleotide Run
Hearts were washed in ice-cold phosphate-buffered saline (PBS), homogenized in 6% trichloroacetic acid, centrifuged and extracted with water-saturated inhalation anaesthetic. The aqueous bodily structure was transferred and void dried, and the orb was resuspended in sodium rayon memory device for cAMP and cGMP enzyme immunoassay (Amersham Pharmacia Biotech).