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Figure 1 | BMC Plant Biology

Figure 1

From: Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

Figure 1

Zera-DsRed expression in N. benthamiana leaves and protein bodies isolation. (a) SDS-PAGE analysis and immunoblot of proteins extracted from N. benthamiana leaves co-infiltrated with Zera-DsRed and the silencing suppressor HcPro. Leaf tissue was collected at 7 days post infiltration (dpi). The Coomassie blue-stained gel (left panel) was loaded with 15 ÎĽg of total protein extracted from both, Zera-DsRed transformed leaf tissue (lane 1) and untransformed leaf tissue (lane 2). The gel for the immunoblot was loaded with 1 ÎĽg of of total protein extracted from Zera-DsRed transformed leaf tissue (lane 1) and untransformed leaf tissue (lane 2). Immunoblot was labelled with anti-R8 antibody (dilution 1:8000). Arrows indicate Zera-DsRed band and arrowheads in immunoblot indicates large oligomers of Zera-DsRed. (b) Confocal microscopy image of induced protein bodies (red) in epidermal cells transformed with Zera-DsRed and collected at 7dpi. (c) Immunoelectron microscopy of a thin section of leaf cell transformed with Zera-DsRed. Zera-DsRed protein inside PB was labelled with anti-R8 antibody- protein A conjugated with gold particles (10 nm). PB: Protein body; ch: chloroplast; M: mitochondria, scale bar: 200 nm (d) Protein analysis by SDS-PAGE followed by silver staining of the various fractions of the PBs isolation by density gradient. H is the homogenate of the leaf tissues loaded in the gradient, S is the supernatant after centrifugation, F1- F6 are the interphase fractions in the increasing densities and P is the pellet. Equivalent volumes were loaded in lanes F1-F6 and P, in H and S only one-third equivalent was loaded. (e and f) Immunoblot of the gradient fractions using anti-R8 (e) and anti-DsRed (f) antibodies. Arrows indicated Zera-DsRed protein present in the homogenate and enriched in the dense F4 fraction. (g) Immunoelectron microscopy of a thin section of isolated Zera-DsRed PB labeled with anti-R8 antibody-Protein A conjugated to gold particles (10 nm), arrows indicated ER membranes with ribosomes, scale bars 200 nm. (h-j) PBs isolated from leaves co-infiltrated with Zera-DsRed and ECFP-SQS1 were monitored by confocal microscopy. (h) Image of cyan fluorescent label of ER-membrane protein SQS1. (i) Fluorescent label of isolated Zera-DsRed PBs (red) and (j) picture shows the merged fluorescent signals and illustrated the presence of membrane protein ECFP-SQS1 surrounding isolated PBs. Scale bar: 2 ÎĽm.

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