Pro q diamond phosphoprotein staining

To examine the effect of β‐adrenergic‐dependent phosphorylation of thin and thick filament proteins, Pro‐Q Diamond phosphoprotein staining (Invitrogen, Grand Island, NY) was employed following the manufacturer's instruction. Total cardiac muscle proteins were resolved on 14% SDS‐polyacrylamide gels as above. The SDS‐gel was fixed in 50% methanol and 10% acetic acid overnight with a change after the first 45 min. After washing in deionized water for three changes of 10 min each, the gel was stained with shaking in Pro‐Q Diamond reagent for 90 min in a dark box. Destaining was performed in 20% acetonitrile, 50 m·mol·L⁻¹ sodium acetate, pH 4.0, for three changes of 30 min each in a dark box. The gel was then washed twice with deionized water for 5 min each in a dark box and scanned on a Typhoon 9410 fluorescence scanner (GE Healthcare, Wauwatosa, WI) with excitation at 532 nm and recording the emission at 560 nm to reveal phosphorylated proteins. The same gel was then stained with Coomassie Blue R‐250 to visualize the total protein profile.

A LV myocardial tissue sample (20 mg) was first handled similarly as for cardiomyocyte isolation, then homogenized in a sample buffer (containing 8 M urea, 2 M thiourea, 3% sodium dodecyl sulfate [SDS], 75 mM DTT, 0.05 M Tris-HCl [pH 6.8], 40 µM leupeptin, 10 µM E64, 10% glycerol, brome-phenol blue). After centrifugation (16 000 g for 5 minutes) protein concentration was measured from the supernatant with a dot-blot based method, where different dilutions from bovine serum albumine served as standard. Protein concentration of the samples was adjusted to 1 mg/mL. Polyacrylamide gels (2%, 4%, and 15% agarose-strengthened) were used to separate titin, MyBP-C, and TnI, respectively. Phosphorylation status of myofilament proteins was assessed by Pro-Q^® Diamond phosphoprotein staining (Invitrogen, Eugene, OR, USA), while protein composition was visualized by Coomassie blue (Reanal, Budapest, Hungary). Separation and identification of myofilament proteins were carried out based on their molecular weight and compared to those of a molecular weight standard (marker 4.6 kDa – 300 kDa).

We analyzed protein phosphorylation using a 12% SDS-PAGE with Pro-Q Diamond phosphoprotein staining (Invitrogen, Eugene, OR, USA). SYPRO Ruby (Invitrogen, Eugene, OR, USA) staining was used to estimate the total amount of protein. Protein samples and gel staining were prepared according to the manufacturer's manual. The gel was scanned on the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA), and band densities were determined with Image Lab 5.2.1 software (Bio-Rad, Hercules, CA, USA). A level of protein phosphorylation was expressed as a ratio of the Pro-Q Diamond intensity to the SYPRO Ruby intensity.

Sarcomeric proteins were obtained using the suspension of single cardiomyocytes. For in vitro motility assay experiments, cardiac myosin was extracted from cardiomyocyte suspensions according to Margossian and Lowey [61 ] with modifications. F-actin and troponin were obtained from the bovine left ventricle [62 ,63 ]. Human a-tropomyosin (Tpm) was expressed in BL21(DE3) bacterial cells [64 (link)]. Regulated thin filaments (RTF) were reconstituted from F-actin, troponin, and Tpm in a flow chamber.
Protein phosphorylation was analyzed using a 12% SDS-PAGE with Pro-Q Diamond phosphoprotein staining (Invitrogen, Eugene, OR, USA). SYPRO Ruby (Invitrogen, Eugene, OR, USA) staining was used to estimate the amount of total proteins. Protein samples and gel staining were prepared according to manufacturer’s manual. The gel was imaged on the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA), and band densities were determined with Image Lab 5.2.1 software (Bio-Rad, Hercules, CA, USA). A level of protein phosphorylation was expressed as a ratio of the Pro-Q Diamond intensity to the SYPRO Ruby intensity.

For in vitro motility assay experiments, cardiac myosin was extracted from LA and RA cardiomyocytes according to [24 ] with modifications. F-actin was obtained from the bovine left ventricle [25 ].
Protein phosphorylation was analyzed using a 12% SDS-PAGE with Pro-Q Diamond phosphoprotein staining (Invitrogen, Eugene, OR, USA). SYPRO Ruby (Invitrogen, Eugene, OR, USA) staining was used to estimate the total amount of protein. Protein samples and gel staining were prepared according to the manufacturer’s manual. The gel was scanned on the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA), and band densities were determined with Image Lab 5.2.1 software (Bio-Rad, Hercules, CA, USA). A level of protein phosphorylation was expressed as a ratio of the Pro-Q Diamond intensity to the SYPRO Ruby intensity.