Fig. 4
SAG-SCFβ-TrCP promotes poly-ubiquitylation of PHLPP1 and DEPTOR: a & c The in vivo ubiquitylation assay: The 293 cells were transfected with indicated plasmids and lysed under denatured condition (6 M guanidinium solution), followed by Ni-bead pull-down. Washed beads were boiled and subjected to IB for PHLPP1 (a) or DEPTOR (c). b & d The in vitro ubiquitylation assay: SAG-CUL1 E3 was prepared by FLAG-conjugated beads IP using 293 cells transfected with SAG, along with CUL1. PHLPP1 or PHLPP1(4A) was prepared by transfecting HA-PHLPP1 or HA-PHLPP1(4A) into 293 cells, followed by HA-conjugated beads IP and 1× HA peptide elution (b) or DEPTOR or DEPTOR(3A) was transfected into 293 cells, followed by FLAG-conjugated beads and 3× FLAG peptide elution (d). SCF E3 and substrates (PHLPP1 or DEPTOR) or their mutants were added, respectively, into a reaction mixture containing ATP, ubiquitin, E1 and E2, followed by constant mixing for 60 min. The reaction mixture was boiled with loading buffer and then loaded onto SDS-PAGE gel for IB using PHLPP1 (b) or DEPTOR (d) Ab