Ab207399

To detect the phosphorylation status of S6K, 50 mg plant aerial tissue was used for protein extraction using 1.5 mL extraction buffer of 1 × PBS, pH 7.4, containing 250 mM sucrose, Protease Inhibitor Cocktail (Sigma-Aldrich, P9599) and PhosSTOP phosphatase inhibitor (Roche, 4906845001). Three times of centrifugation, 1 k × g for 5 min, 14 k × g for 5 min and 135 k × g for 30 min, were conducted to separate the soluble proteins. The supernatant from the last centrifugation was separated, concentrated to 200 μL using an Amicon Ultra centrifugal unit (Millipore, UFC501024), and then mixed with 40 μL 6 × Laemmli buffer. Proteins were denatured by incubation at 95°C for 10 min. Protein samples were separated on 15% SDS-PAGE with 8M urea and blotted to PVDF membranes (Bio-Rad, 1620177). Blots were blocked with 5% milk for 1 hr at room temperature. Blots were incubated with primary antibodies of either anti-S6K (Agrisera, AS12 1855) or anti-S6K-phosphorylated (Abcam, ab207399) overnight at 4°C and subsequently with secondary HRP conjugated goat anti-rabbit antibody (Sigma-Aldrich, A0545) for 1 hr at room temperature.

At least 120 quiescent seedlings per sample were collected and frozen in liquid nitrogen. Protein was then extracted from the plant tissue in 100 mM MOPS (pH 7.6), 100 mM NaCl, 5% SDS, 0.5% β-mercaptoethanol, 10% glycerin, 2 mM PMSF, and 1x PhosSTOP phosphatase inhibitor (Sigma-Aldrich). S6K-pT449 was detected by Western blot using a phosphospecific antibody (ab207399, AbCam) and an HRP-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immuno Research, no. 111-035-003). S6K levels were detected by Western blot using a custom monoclonal antibody described in Busche et al., 2020 (link). Total protein was visualized after transfer using Ponceau S red staining. Western blot images were scanned, converted to grayscale, and adjusted for contrast and brightness using ImageJ.