Tc287

Detection of specific proteins among the experimental series required a proper sample preparation, in which, for each specimen, the Laemmli buffer 2X (S3401; Sigma-Aldrich) was mixed with an equal protein amount before heat denaturation (5 min at 95 °C). Subsequently, loading a protein range from 15 to 30 µg, electrophoresis was performed in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). After moving proteins to the nitrocellulose membrane (Amersham Protran Premium; Cytiva, Marlborough, MA, United Stetes), foils were firstly blocked for one hour in no-fat milk (5% w/v) (A0830; PanReac Applichem, Chicago, IL, USA) and then incubated overnight at 4 °C with specific primary antibodies. The next morning, HRP secondary antibodies, recognizing the related primary species, were applied to the membrane for 1 h at room temperature. Finally, chemiluminescence was detected with the Chemi Doc XRS (Bio-Rad, Hercules, CA, USA) instrument using liteablot chemiluminescent as a substrate (EMP011005; EuroClone). Each incubation step was preceded and followed by three washes in TBS plus 0.05% Tween-20 (TC287; HIMEDIA, Mumbai, India) (T-TBS).

Approximately 10–30 µg of cell protein extract and 5–50 µg of serum or urine proteins were loaded in 10% acrylamide gels. Later, proteins were transferred onto a nitrocellulose membrane (GEH10600008, Cytiva, Marlborough, MA, USA) by Mini Trans-Blot (Bio-Rad Laboratories, Hercules, CA, USA). In order to reduce both noise and aspecific bond, potential free spots on nitrocellulose film were covered with non-fat milk (5% w/v) (A0830, PanReac Applichem, Chicago, IL, USA) for one hour. Anti-PKA-Cα (sc-903, Santa Cruz Biotechnology, Dallas, TX, USA) was subsequently incubated overnight at 4 degrees. The next day, HRP-conjugated goat anti-rabbit (#7074, Cell Signaling Technology) was added to the membranes for one hour at room temperature. Washing with TBS Tween-20 (TC287, HIMEDIA, Mumbai, India) preceded and followed each single procedure. Lastly, using enhanced chemiluminescence reagent (E-IR-R301, Elabscience, Houston, TX, USA), immunoblotting bands were detected and acquired by Chemi-Doc XRS (Bio-Rad Laboratories).

Depending on the target protein, an amount of 10–30 μg of total extracts was loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for each sample. Subsequently, sample proteins were transferred onto nitrocellulose membranes (GEH10600008; Amersham) by the Mini Trans-Blot system (Bio-Rad Laboratories). After washing in tris-buffered saline (TBS) supplemented with 0.05% Tween 20 (TC287; HIMEDIA), films were blocked 1 h in 5% no-fat dry milk (A0530; AppliChem) aimed at covering potentially free spots into the nitrocellulose membrane. Incubation overnight at 4°C has been chosen for primary antibody binding. In the following days, horseradish peroxidase (HRP)-conjugated secondary antibodies, reacting against the related primary species, were applied to the membrane for 1 h at room temperature. Each incubation step was preceded and followed by three 5-min rinses in T-TBS. Finally, protein-related light signals were acquired by ChemiDoc™ (Bio-Rad Laboratories) using the enhanced luminol-based chemiluminescent substrate (E-IR-R301; Elabscience) as a detection system for HRP.