Immunolocalization of SYT2.

Bars = 10 µm. (A) Protein gel blot of SYT2 in wild-type (WT), syt2-1, and SYT2-GFP overexpressing plants (SYT2-GFP1 and SYT2-GFP2 are different lines). The blots were probed with polyclonal anti-SYT2 (Right) and monoclonal anti-GFP (Left) antibodies to detect the SYT2 and GFP-tagged proteins, respectively. The expected sizes of the proteins are indicated. The bottom images show Coomassie Brilliant Blue staining (CBB) as loading controls. (B) RT-PCR analysis of SYT1 and SYT2 in syt2-1 and wild-type plants. Actin served as a control. (C and D) Localization of SYT2 in root cells of wild-type plants. Root tissues of Arabidopsis grown on ½ MS solid medium for 3–4 days were prepared for immunolabeling with normal rabbit serum (as a control) (C), or anti-SYT2 antibody as the primary antibody (D) and fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG as the secondary antibody. (E) Double-labeling with anti-SYT2 and anti-GFP antibodies in root cells of SYT2-GFP-overexpressing seedlings. Anti-SYT2 and anti-GFP antibodies were labeled with tetramethylrhodamine-5-isothiocyanate (TRITC)-labeled anti-rabbit IgG and FITC-labeled anti-rat IgG, respectively. Arrows indicate the overlap of green and red fluorescent signals. (F) Double-labeling with anti-SYT2 and anti-GFP antibodies in root cells containing Golgi marker ST-YFP. Anti-SYT2 and anti-GFP antibodies were used as in (E). Arrows indicate the overlap of green and red fluorescence signals. (G–L) Immuno-gold labeling and electron microscopic observation showed that SYT2 was located on Golgi apparatus in root tip cells of Arabidopsis. (G and H) Electron microscopic observation showed that SYT2 was located mainly on Golgi apparatus in root tip cells of wild type plants. (H) High-magnification image of Golgi apparatus in the inset in (G). (I and J) Immuno-gold labeling of Golgi apparatus in root tip cells of SYT2-overexpressing plants. (J) High-magnification image of Golgi apparatus in the inset in (I). (K and L) Control section, incubated with the secondary antibody alone, did not show gold particles on Golgi apparatus. G: Golgi apparatus. Bars: 2 µm (G, I,); 50 nm (H, J, K, L).