Anti his6

SDS-PAGE as previously described^{21 (link)} performed using a 10% polyacrylamide resolving gel with a 4% stacking gel (Bio-Rad Mini-Protean). Western blotting, made using primary potato antibodies for P1-G6PDH, P2-G6PDH and Cy-G6PDH isoforms^{13 (link)} and Anti-His6 (Roche). Potato antisera were previously proven to react with and discriminate the different G6PDH isoforms^{20 (link),21 (link),53 (link)}. After incubating the membrane with secondary antibodies and cross-reacting polypeptides stained by enhanced chemiluminescence.

Total yeast protein extracts were prepared as previously described [58 (link)]. Cultures were grown to an OD₆₀₀ of around 0.8 and protein extracts were prepared from an equivalent of one OD₆₀₀ of cells. Western blot analysis was carried out according to standard protocols. The following primary antibodies were used in this study: mouse monoclonal anti-FLAG (1:2’000–1:10’000; Sigma), anti-GFP (1:2’000; Roche), anti-HA (1:3’000; BAbCO), anti-His₆ (1:500; Roche), and anti-Rpl3 (1:5’000; J. Warner, Albert Einstein College of Medicine, New York); rabbit polyclonal anti-Adh1 (1:50’000; obtained from the laboratory of C. De Virgilio, University of Fribourg), anti-CBP (1:15’000; Open Biosystems), anti-Rpl5 (1:5’000; S.R. Valentini, São Paulo State University, Araraquara), and anti-Rps3 (1:20’000; M. Seedorf, ZMBH, University of Heidelberg, Heidelberg). Secondary goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Bio-Rad) were used at a dilution of 1:10’000. For detection of TAP-tagged proteins, the Peroxidase-Anti-Peroxidase soluble complex was used at a dilution of 1:20’000 (Sigma). Immobilized protein-antibody complexes were visualized by using enhanced chemiluminescence detection kits (Amersham ECL, GE Healthcare; PicoDetect, Applichem; WesternBright Sirius, Advansta).