Western Blot Analysis of Protein Interactions

All experiments were performed as described previously (Kolonko et al., 2020 (link)). Briefly, samples obtained during the pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich). The membranes were blocked with milk powder (Milchpulver, blotting grade, Roth), then incubated overnight with specific primary anti-GFP polyclonal antibodies (1:5,000, cat. no. SAB2702197, Sigma-Aldrich) or anti-14-3-3 antibodies (1:5,000, cat. no. AB9748-I, Millipore), followed by the incubation with secondary horse anti-mouse antibodies (for anti-GFP, 1:10,000, cat. no. PI-2000, Vector Laboratories) or goat (for anti-14-3-3, 1:10,000, Sigma-Aldrich, cat. no. A 6154) anti-mouse antibodies coupled to horseradish peroxidase. Signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescent kit (Thermo Scientific™). Finally, the membranes were exposed to Kodak BioLight film. The MM of each fusion protein was calculated based on aa sequences using the ProtParam server (Gasteiger, 2019 ) (YFP-Met-FLAG 107 kDa, YFP-Gce-FLAG 105 kDa, YFP-MetC-FLAG 51.4 kDa, YFP-GceC-FLAG 64 kDa, CFP-Ftz-F1 LBD 55 kDa, and CFP-14-3-3 57 kDa).