Anti 14 3 3 antibody

For in vitro pull-down assays, all recombinant proteins were purified from E. coli DE3 strains. Briefly, GST-tagged SOS2-JD protein retained on the beads incubated with His-tagged PKS5 in kinase buffer with ATP at 30 °C for 30 min. After removed His-tagged PKS5 by washing three times with PBS buffer, GST-tagged SOS2-JD protein were incubated with His-tagged 14-3-3λ in binding buffer (2 mM DTT, 10 mM MgCl₂ and 20 mM Tris–HCl, pH 7.2) at 4 °C for 3 h, and then the beads were wash for three times with PBS buffer. The pulled-down proteins were detected by immunoblot analysis using an anti-14-3-3 antibody (Santa Cruz SC-33752, 1/2000).
For in vivo Co-immunoprecipitation assays, stable transgenic plants were used to detect the interaction between 14-3-3 proteins and SOS2/PKS5 and the phosphorylation of SCaBP8^Ser237inplanta. Total proteins were extracted from Arabidopsis using IP buffer (10 mM Tris, pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail; Roche). After removed cellular debris, the supernatant was incubated with anti-C-Myc agarose (Sigma-Aldrich) at 4 °C for 3 h, and then the beads were washed for five times with IP buffer. The associated proteins were analyzed by immunoblots and detected with anti-C-Myc (CWBIO 01217/10153, 1/3000) and anti-P-SC8 (made by AbMart, 1/2000) antibodies.