Figure 6.

IDRs contribute to EDEM1 half-life but not to the ERjd5 association. A, left panel, FLAG-tagged EDEM1 ΔIDR(N)/(C), and MLD were transfected into HEK293T. Proteins were radiolabeled for 30 min with [³⁵S]Cys/Met, and cells were lysed in MNT buffer. Time points were collected at 0, 2, and 4 h after the pulse. Proteins were resolved by reducing 8% SDS-PAGE. Right panel, the amount of EDEM1 protein remaining at 2 and 4 h was quantified and normalized to the starting material (0 h) and averaged from three independent experiments. The starting material includes both bands of the observed doublet. Statistical significance between MLD, EDEM1 ΔIDR(N), or EDEM1 ΔIDR(C) and EDEM1(FL) at 4 h was determined by an unpaired t test; the measurement designated ** for MLD has a p value of 0.005, and that designated * for EDEM1 ΔIDR(C) is 0.012. Error bars represent mean ± S.E. B, FLAG-tagged EDEM1(FL), EDEM1 ΔIDR(N), EDEM1 ΔIDR(C), and MLD and empty plasmid (Mock) were coexpressed with ERdj5-MYC in HEK293T cells. Cells were lysed in HBS buffer containing CHAPS. 10% of the lysate was collected for whole-cell lysate (WCL), and 40% was collected for anti-FLAG and anti-MYC immunoprecipitation (IP). Proteins were resolved by 8% reducing SDS-PAGE and immunoblotted against MYC, FLAG, and glucosidase II (GlucII). Gels are representative of three independent experiments.