Figure 4.

Visualizing the candidate protein interactors of NtPhyt. Proteins biotinylated in NtPhyt-TurboID- or SP-TurboID-producing leaves during the incubation of leaf samples with 200 μM biotin for 5 h (Phyt and SP, respectively) were isolated from the respective intracellular fractions (obtained from equal 5 g amounts of leaf tissues) by extraction in the presence of 0.5% dodecyl maltoside. Upon ammonium sulphate (AS) fractionation, biotinylated proteins precipitated with 50% AS and 70% AS were separated by affinity chromatography on streptavidin magnetic beads and analysed by SDS gel electrophoresis. (a) On-blot detection of biotinylated proteins using streptavidin-HRP. One % of the affinity purified protein samples in 4 μL aliquots were loaded. (b) The rest of the samples, after acetone precipitation, were dissolved in 45μL of SDS-containing buffer and analysed by SDS gel electrophoresis. Coomassie blue staining of the 6–16% gradient polyacrylamide gel. Arrows with numbers indicate the bands chosen for protein identification by mass spectrometry (MS) analysis. M, molecular weights of the protein markers.