Fig. 1

CRISPR screen identifies SPOP as a bona fide E3 ligase for PDK1. A A schematic illustration for the plenti-hygro-CMV-DsRed-IRES-EGFP-PDK1 reporter system. B Flow-chart of genome-scale library screening using CRISPR/Cas9 system. C The selected reporter cells were sorted by flow cytometry. D Identification of top candidate sgRNA genes using a scatter plot after high-throughput sequencing. E Stable expression of DsRed/EGFP-PDK1 HEK293T cells were infected with lenti-CRISPR sgRNAs targeting control or SPOP for 7 days and then analyzed by flow cytometry. F Immunoblot (IB) analysis of WCL derived from C4-2 and 22Rv1 cells. Where indicated endogenous SPOP was knockout by using CRISPR/Cas9. G IB analysis of WCL derived from Spop^−/− and counterpart MEFs. H IB analysis of WCL derived from RV1 and DU145 cells transfected with indicated lentiviral shRNAs. The transfected cells have been selected with puromycin (1 μg/ml) for 72 h to eliminate the uninfected cells before harvested. I, J IB analysis of control and SPOP knockdown PC3 cells. Where indicated cells were treated with CHX (100 μg/ml) for the indicated time points before harvested. PDK1 protein abundance in (I) was normalized and quantified (H) (mean ± SD, n = 3) (t test), *P < 0.05, **P < 0.01. K IB analysis of WCL and His pulldown products derived from HEK293T cells transfected with indicated constructs. Cells were treated with MG132 (10 μM) for 12 h before harvested. L In vitro ubiquitination assays were performed with commercial E1, E2 and Ub proteins, bacterially purified His-PDK1, as well as SPOP purified from SPOP-overexpressing HEK293T cells. The reaction products were subjected to IB analysis