Co-localization of RPG with ER and TGN/EE markers but not Golgi or MVB markers.

Protein subcellular localizations were analyzed by confocal microscopy of proteins co-expressed in N. benthamiana leaf cells. (A) Fluorescence from GFP-RPG (green) and (A′) the ER marker Sec12-PHB-mCherry (magenta) was imaged and the merged image (A″) shows that the punctate expression of GFP-RPG occurs in specific regions that are associated with fluorescence from Sec12-PHB-mCherry. A plot (A‴) of fluorescence intensities of GFP-RPG and Sec12-PHB-mCherry fluorescence in a region of interest (white line in A″) shows that GFP-RPG colocalized with Sec12-PHB-mCherry, whereas other regions of Sec12-PHB-mCherry do not show GFP-RPG localization. (B) Fluorescence from the TGN/EE marker HAP-13-GFP (green) and (B′) from mCherry-RPG colocalized based on the merged image (B″) and (B‴) the plot of fluorescence intensities of HAP-13-GFP and mCherry-RPG in the area of interest (marked with a line in B″). (C) Fluorescence from GFP-RPG and (C′) the MVB marker ARA6-mCherry. The punctate expression of GFP-RPG (green) and the punctate localization of foci of the multivesicular body (MVB) marker ARA6-mCherry SR2 did not colocalize based on the merged image (C″) and the enlargement of it (C‴). (D) The green GFP-RPG fluorescence and (D′) red fluorescence from mRFP fused to the-vascular sorting peptide 2 (mRFP-VSR2) did not colocalize based on the merged image (D″) and the enlargement of it (D‴). (E) The fluorescence from the Golgi marker CD3-963-GFP (green) and (E′) mCherry-RPG (magenta) did not colocalize based on the merged image (E″) and the enlargement of it (E‴). (F) The RPG-CERBERUS BiFC construct (yellow) and (F′) the ER marker Sec12-PHB-mCherry (magenta) colocalized based on the merged image (F″) and the Plot (F‴) showing fluorescence intensities of Venus and Sec12-PHB-mCherry in regions of interest (white line in F″). Scale bars: 25 μm (A-F). (TIF)