Mini protean

Proteins were separated on SDS–Page in a Mini Protean apparatus (Bio-Rad, Hercules, CA, USA) [54 (link)] and then electroblotted on PVDF membrane with the Trans–Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Anti-STRP [7 (link)] 1:10,000, anti-actin (Agrisera, Vännäs, Sweden) 1:5000, and anti-GFP (Santa Cruz Biotechnology, Inc., Heidelberg, Germany, DE) 1:1000 dilutions were used. Protein detections were performed by incubating the membrane with horseradish peroxidase–conjugated anti-rabbit secondary antibody (1:5000) or anti-mouse secondary antibody (1:5000) from Bio-Rad.

BN-PAGE was performed as previously reported [25 (link)]. Briefly, polyacrylamide native gradient gels (3–9%) were prepared in Mini-PROTEAN (Bio-Rad, California, USA) with 1.5 mm spacers. Protein samples, prepared as previously described, were loaded in each lane. Anode buffer (25 mM Imidazole pH 7.0) and Cathode Buffer Blue (50 mM Tricine, 7.5 mM Imidazole, 0.02% Coomassie Blue G-250) were used as running buffers and the electrophoresis was performed at 6 mA at 4 °C. After the tracking line of Coomassie Blue G-250 dye reached half of the gel, cathode buffer blue was removed and substituted by cathode buffer (50 mM Tricine, 7.5 mM Imidazole). The electrophoresis continued at 6 mA at 4 °C and was stopped when the blue line had left the bottom of the gel. Finally, proteins were transferred to PVDF membranes (Millipore, Burlington, Massachusetts, USA) for immunoblot analysis, as described below.

The protein extracts were subjected to SDS–PAGE using a Mini Protean apparatus (Bio-Rad Laboratories, PA, USA) and transferred to polyvinylidenedifluoride (PVDF) membranes. For heart and kidney tissue, 40 μg of protein homogenates were loaded onto the polyacrylamide gel whereas for HEK293T cells 25 μg were used.
To reduce non-specific antibody binding, membranes were incubated for 2 h at room temperature in bovine serum albumin (BSA)-based blocking buffer. The membranes were then exposed to primary antibodies overnight at 4°C. Only those antibodies recommended for WB (Fig 1) were tested. Based on the described reactivity we used AAR-013 (1:5000) and sc-135063 (1:1000), for detection of the MasR present in mouse tissues, while AAR-013, sc-135063 and sc-54682 1:1000 were used for detection of the human receptor expressed in HEK293T cells. Anti c-myc tag antibody (Cell Signaling Technology Inc.; cat. #2272) diluted 1:1000 was used to detect the c-myc tagged MasR. Anti-β-tubulin (Abcam Inc.; cat. #Ab6046) diluted 1:5000 was used as a protein loading control. After incubation for 1 h at room temperature using a 1:20000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibody, sc-2004 (anti-rabbit) and sc-2020 (anti-mouse) (Santa Cruz Biotechnology, CA, USA), reaction products were revealed by enhanced chemiluminescence (ECL Plus, Pierce, Rockford, IL, USA).

Total leaf proteins were extracted in Laemmli [98 (link)] solubilizing buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/V) SDS, 2% (w/V) DTT, 8.7% (w/V) glycerol) and further solubilized at room temperature for 30 min. Samples containing about 10 µg proteins and 0.001% (w/V) bromophenol blue were applied per lane. Polypeptides were separated according to Laemmli [98 (link)] by applying 10–18% gradient polyacrylamide gels containing 8.7% (w/V) glycerol using a MiniProtean apparatus (BioRad, Hercules, CA, USA) with a constant current of 20 mA per gel at 6 °C for 2 h.

The protein samples were heated at 95°C for 10 minutes with an equal volume of 2× sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol) and separated by SDS-PAGE in 12% gel [50] (link). For immunoblotting, the separated proteins were transferred to nitrocellulose membranes according to the instructions for the electroblotting apparatus (Mini-PROTEAN, Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked for 1 hour in TTBS (TBS with 0.05% Tween-20) with 1% BSA, washed with TTBS, and incubated overnight at 4°C with purified Abs diluted 1∶1000. After washing with TTBS, the membranes were incubated for 1 hour with goat anti-rabbit Abs conjugated with peroxidase (Bio-Rad Laboratories) diluted 1∶4000 in TTBS, consequently washed with TTBS and TBS, and the peroxidase reaction was performed with 4-chloro-1-naphthol (Sigma-Aldrich, St Louis, MO, USA) as a substrate.