Anti 14 3 3

Anti-Myc (Cat# M4439 RRID: AB_439694) and anti-FLAG (Cat# F3165 RRID: AB_259529) were purchased from Sigma-Aldrich (USA). Anti-H3 (Cat# AS10 710 RRID: AB_10750790), anti-RbcL (Cat# AS03 037–200 RRID: AB_2175288) and anti-14-3-3 (Cat# AS12 2119 RRID: AB_2619715) were from Agrisera (Sweden). Anti-GFP (Cat# MMS-118P-200 RRID: AB_10063778) was from Covance (USA).

[³⁵S]Met-labeled preproteins synthesized in RRL (Promega, United States) were incubated with rotation for 1 h at RT in the presence of preimmune sera (GenScript, United States), anti-14-3-3 (a gift from Prof. Carol MacKintosh, University of Dundee) or anti-Hsp70 antibodies (Catalog no.: AS08 371; Agrisera, Sweden) in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 0.2% Triton X-100, pH 7.5) (Dessi et al., 2003 (link)). The antibodies were prepared against KLH-conjugated synthetic peptide conserved in the five cytosolic Hsp70 proteins of Arabidopsis thaliana (Sung et al., 2001 (link)). The bound proteins were pulled down using Protein-A Sepharose beads (Thermo Scientific, United States) and washed by reaction buffer containing 250 mM NaCl four times. After the supernatant was discarded, the Protein-A Sepharose beads (Invitrogen, United States) was resuspended in Lammeli sample buffer and denatured at 95°C for 5 min. The immunoprecipitated proteins were analyzed in SDS-PAGE gel and detected by autoradiography.