Western Blot analysis of samples from yeast whole cell extract expressing the Arabidopsis thaliana subunit E1. Primary antibodies have been used at 1: 1: 000. Secondary antibodies at 1: 3 000. The load per well was 10 ?g of total protein. Samples were separated on 10 % gel in Tris glycine SDS buffer. The proteins were transferred to a membrane (Schleicher & Schuell), blocked with 5% dry milk in Tris-buffered saline before incubation for 2 hours up to over night at RT with the primary antibody. A series of washes with TTBS was followed by incubation with a secondary antibody from).

Western Blotting: 1 - molecular weight markers (Precision Plus Protein Standards, Dual Color, Bio-Rad, 10?l); 2 -Arabidopsis thaliana leaf extract (25 ?g) Experimental Conditions: Protein was separated on a 12% SDS-PAGE gel at 200 V for approximately 40 min using the mini-Protein 3 cell (Bio-Rad). Protein was transferred to PVDF at 100 V for 1h using the mini-TransBlot cell (Bio-Rad). Blocked overnight at 4ºC in 5% non-fat milk dissolved in 1xPBST. Incubated with anti-V-ATPase at 1:2 000 for 2h at RT. After several washes in 1xPBST, blot was incubated with goat anti-rabbit IgG-alkaline phosphatase conjugate (Bio-Rad) at 1:3000 for 1h at RT. Blot was developed with NBT/BCIP (Molecular Probes). Courtesy: Ms Jaimie Schnell, PhD candidate

Standard IF protocol for plant material has been used including slight fixation with formaldehyde followed by washing and incubation with primary and secondary antibodies conjugated to fluorescent dyes. Green dye visualization of anti-V-ATPase antibody (Alexa 488 Molecular Probes), red – anti-tubulin antibody.