O2 is rate limiting for Skp1 hydroxylation. The modification status of Skp1was analyzed as a function of O2 level. Whole cell extracts (Ax3 or phyA–) harvested after 24 h of submerged development were subjected to SDS-PAGE and Western blotting. Blots were sequentially probed with (A) pAb UOK87, which preferentially (but not exclusively) recognizes unmodified Skp1 , (B) mAb 4E1, which recognizes all Skp1 isoforms, and (C) anti-actin, as a loading control. Skp1 migrated predominantly as 2 bands: an upper glycosylated band, and a closely-spaced lower non-glycosylated band (preferentially labeled by pAb UOK87) that was partially contaminated by glycosylated Skp1. (D) The levels of glycosylated and unmodified Skp1 were quantitated by densitometry of mAb 4E1-labeled blots as described in Methods, and the fraction of total Skp1 that is unmodified is plotted as a function of O2 level in the atmosphere. Data are from 5 independent experiments and are reported ± SEM. (E) The number of spores formed after 3 d for the trial shown for panels AC is shown.
Xu et al. BMC Developmental Biology 2012 12:31 doi:10.1186/1471-213X-12-31