Phospho ser thr pka substrate antibody 9621

RyR1 was immunoprecipitated from 400 μg of mouse skeletal mixed membrane preparations using an anti‐RyR antibody (34C; Abcam, Cambridge, UK) and Protein G Dynabeads (Life Technologies, Oslo, Norway) by overnight incubation at 4°C with continuous mixing in 0.4 ml of homogenization buffer [20 mm HEPES, 150 mm NaCl, 5 mm EDTA, 20% (v/v) glycerol, protease inhibitors (Roche Diagnostics Limited, Burgess Hill, UK), Triton‐X 0.5%, pH 6.8]. Protein immunocomplexes were separated magnetically (DynaMag‐2 magnet; Thermo Fisher) and beads were washed three times with homogenization buffer. For PKA phosphorylation of RyR1, beads were incubated with 1 U of the catalytic subunit of PKA (Sigma‐Aldrich) per μg protein for 10 min at 37°C in a solution containing 50 mm HEPES, 16 mm Tris, 5 mm Mg²⁺ and 5 mm NaF (pH 7.2). After PKA treatment, the supernatant was removed by magnetic separation and samples were resuspended in 50 μl of Laemmeli sample buffer containing 5% β‐mercaptoethanol and incubated at 95°C for 5 min. Samples were then used for western blotting as described previously (Carter et al. 2011). Immunoblots were probed with anti‐RyR antibody (34C; dilution 1:1000) and Phospho‐(Ser/Thr) PKA Substrate Antibody #9621 (dilution 1:1000; Cell Signaling Technology, Leiden, The Netherlands). RyR1 protein and phosphorylation levels were quantified by densitometry.

Lysates from transfected MCF-7 cells were separated by SDS-PAGE and immunoblotted as previously described [17 (link)]. Antibodies against IRS-1 (sc-560) and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibody against FLAG tag (F7425) was from Sigma Aldrich (Co., St. Louis, MO, USA). Antibodies against AKT, phosphorylated AKT (Ser473), Erk1/2, phosphorylated Erk1/2 (Thr202/Tyr204; D13.14.4E), and phospho-(Ser/Thr) PKA substrate antibody (9621) were from Cell Signaling Technologies (Beverly, MA, USA). Antibody against PI3K-p85α was from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). For immunoprecipitation, cell lysates (1 mg) were precleared with 1 μg of Normal IgG and then incubated with 4 μg of antibodies against p85α^PI3K (SC-1637 Santa Cruz Biotechnology, Inc Santa Cruz, CA, USA) or against FLAG tag (A4596 Sigma Aldrich, Co., St. Louis, MO, USA). Immunocomplexes were precipitated following the manufacturer's instructions and processed for Western blot analysis as described. Image analysis for all gels was performed with ImageJ software using the “Gel Plot” plug-in.