Fig. 5: Formation of membraneless NPH3 condensates is independent of 14-3-3 binding.
a Representative anti-GFP immunoblots following subcellular fractionation of protein extracts prepared from dark-adapted N. benthamiana leaves transiently expressing GFP:NPH3 variants. Expression was driven by the 35S promoter. Proteins in each fraction (7.5 μg) were separated on 7.5% SDS-PAGE gels. It is noteworthy that the total amount of soluble proteins (S) is ~15 times higher as compared to the total amount of microsomal proteins (M) after 100,000 × g centrifugation. b Representative confocal microscopy images of leaf epidermal cells from dark-adapted N. benthamiana transiently expressing RFP:NPH3-4K/A-S744A. Z-stack projection is shown. Expression was driven by the 35S promoter. RFP:NPH3 is shown as control. Scale bars, 25 μm. c Representative confocal microscopy images of leaf epidermal cells from dark-adapted N. benthamiana transiently co-expressing GFP:NPH3-S744A and either MAP:mCherry:SAC1 or MAP:mCherry:SAC1DEAD. Expression was driven by the 35S promoter. Z-stack projection of GFP:NPH3-S744A co-expressed with MAP:mCherry:SAC1 is shown. Single expression of GFP:NPH3-S744A is shown as control. Scale bars, 25 μm. d–f Single-cell time-lapse imaging of RFP:NPH3 condensation induced by GFP laser treatment of transiently transformed and dark-adapted N. benthamiana. Expression was driven by the 35S promoter. The image of time point 0 was taken in the absence of the GFP laser. Z-stack projections from selected time points of blue light treatment (d), fluorescence intensity per body (mean ± SEM) (e), and number of bodies (f) are shown. One representative experiment of five replicates is shown. Scale bars, 25 μm. Experiments a–c were performed at least three times with similar results.
