Fig 5. MAGEL2 and USP7 alter CRY1 stability through ubiquitination-related processes.
A) USP7 stabilizes CRY1 when co-expressed in U2OS cells. U2OS cells were transiently transfected with cDNA constructs encoding V5-USP7 and FLAG-CRY1 in equal amounts. B) In a ubiquitination assay, HEK293T cells were co-transfected with epitope-tagged constructs and treated with MG132 and chloroquine. After 24 h, cell lysates were collected, and an aliquot was retained as input and immunoblotted (IB) to confirm expression of V5-USP7. FLAG-CRY1 was immunoprecipitated (IP) using anti-FLAG beads from the remaining lysate. FLAG-CRY1 was detected in the immunoprecipitate, and ubiquitinated CRY1 (Cry1Ub) was detected by probing the immunoprecipitate with anti-HA antibodies to detect HA-ubiquitin (HA-Ub, smear above molecular weight of CRY1). C) Ubiquitination assay showing effect of co-expression of MAGEL2 (V5-MAGEL2 wild-type (WT), V5-MAGEL2p.R1187C (RC), or V5- MAGEL2p.LL1031AA (LA)) on the ubiquitination of CRY1 (Cry1Ub) in transfected HEK cells. D) MAGEL2 and USP7 have opposing effects on the steady-state abundance of CRY1 in co-transfected cells.