Pageruler plus prestained ladder

The SDS stability assay is based on previously reported assays [35] (link), [36] (link). To 55 µM protein in pH 7 buffer either a 10-fold molar excess of biotin-5-fluorescein or the same volume of pH 7 buffer was added. Due to the high isoelectric point of avidin, A/A2-1, A/A2-B and A/A2-B I109K (pI>9), the proteins (including AVR2, pI = 4) were acetylated by adding a 40-fold molar excess of Sulfo-NHS-acetate (Thermo Scientific) [35] (link). The protein sample was divided into equal aliquots and incubated for 20 min at 20, 40, 50, 60, 70, 80, 90, or 100°C. After incubation, the same volume of 2× sample buffer (60 µM Tris-HCl, pH 6.8, 25% glycerol, 0.01% Bromphenol blue) was added, before loading 5 µg of the protein in a 15% acrylamide-bisacrylamide gel. To detect the bound biotin-5-fluorescein, the gels were analyzed under UV in a Molecular Imager Gel Doc XR+ System (BioRad Laboratories, Hercules, CA, USA), before Coomassie Brilliant Blue staining. As a control, proteins were incubated in denaturizing SDS sample buffer (60 µM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% Bromphenol blue, 0.5% β-mercaptoethanol) for 20 min at 100°C. PageRuler Plus Prestained Ladder (Thermo Scientific) was used as molecular weight marker.

Ang II and nebivolol were obtained from Sigma-Aldrich (St. Louis, MO, USA), and L-NAME was obtained from Calbiochem (La Jolla, CA, USA). Antibodies for phospho-mTOR (pSer2448), mTOR, phospho-p70S6K (pThr389), p70S6K, phospho-RPS6 (pSer235/236), and RPS6 were obtained from Cell Signaling Technology Inc. (Boston, MA, USA). ECL substrates for high-sensitivity Western blot detection, Pageruler plus prestained ladder, and SuperScript™ III First-Strand Synthesis System were obtained from Thermo Fisher Scientific (Waltham, MA, USA). TRIzol™ Reagent was obtained from Invitrogen (Waltham, MA, USA), and PowerUp SYBR Green master mix was obtained from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, USA).