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. 2014 Oct 23;14(1):66–80. doi: 10.1074/mcp.M114.044560

Fig. 1.

Fig. 1.

Rate of Skp1 hydroxylation in cells. A, gnt1– cells were transferred to fresh medium and shaken under an atmosphere of the indicated O2 percentage. Metabolic labeling with [35S]Met/Cys commenced at 2.5 h, and cells were harvested at the indicated times and processed for immunoprecipitation using antibodies specific for HO-Skp1 (pAb UOK85) or both HO-Skp1 and Skp1 (pAb UOK77 or mAb 4E1). B–D, time-dependent incorporation of 35S into HO-Skp1 and total Skp1 in cells held at the indicated O2 levels. Data in the same panel are from parallel experiments. Maximal incorporation values into total Skp1 and HO-Skp1 pools after 4 h at 21% O2 were adjusted to 1 to account for variations in antibody pulldown efficiency, and the factor was applied to the parallel low-O2 samples, except for the data in D, which employed the average of factors used for B and C. E, a parallel 4-h sample was subjected to whole cell Western blot analysis using HO-Skp1 (UOK85) and pan-specific (4E1) antibodies to estimate the fraction of Skp1 that was hydroxylated after 6.5 h of exposure to the indicated O2 level. The fraction of Skp1 that was unmodified at steady state in 21% O2 was set at 10% based on the ratio of unmodified to glycosylated Skp1 bands in the wild-type strain Ax3. The unmodified fraction at other O2 levels was derived from the modified fraction via subtraction.