FIGURE 4.

CRL promotes polyubiquitination and degradation of INSL6. A, co-expression of CUL4B resulted in increased amount of multiple slower migrating forms of polyubiquitinated INSL6 in HEK293 cells. Immunoblotting against MYC (Myc-INSL6), HA (HA-CUL4B), and α-tubulin, respectively, were performed on lysates prior to nickel-nitrilotriacetic acid immunoprecipitation. B, immunoblotting against V5 (INSL6-V5), HA (HA-CUL4B), and α-tubulin, respectively, on HEK293 cell lysates, co-transfected with INSL6-V5 and indicated amount of HA-CUL4B plasmid. C, gene silencing by RNAi of DDB1 and RBX1 resulted in reduction of polyubiquitinated INSL6 in HEK293 cells. D, co-immunoprecipitation of DDB1 and INSL6 in HEK293. β-Actin was used as loading control. IB, immunoblotting; IP, immunoprecipitation; Ni-NTA, nickel-nitrilotriacetic acid.