Influence of intramolecular disulfide bridges and ERdj5 on IL-12α degradation.
A, measurement of IL-12α turnover by CHX chase assays. 293T cells were transfected with the indicated IL-12α subunits and incubated with CHX for up to 4 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Hsc70 served as a loading control (top panel). The anti-IL-12α immunoblot signal was normalized to the signal present at the beginning of the chase for the respective IL-12α constructs (bottom panel, n = 4 ± S.E.). Half-lives from exponential fits of the curves (± S.D.) are shown below the graph. MW, molecular weight. B, influence of ERdj5 overexpression on the IL-12α redox state. The amount of HMW and LMW IL-12α species in the absence or presence of ERdj5 overexpression was analyzed (n = 3 ± S.E.; *, p < 0.05). Expression of FLAG-tagged ERdj5 was verified by immunoblotting. C, influence of ERdj5 overexpression on IL-12α degradation. IL-12α turnover by CHX chase assays in the absence (data taken from A) or in the presence of ERdj5 overexpression (n = 3 ± S.E.).