Fig. 2
From: SKP2 promotes breast cancer tumorigenesis and radiation tolerance through PDCD4 ubiquitination
SCFSKP2 is a direct E3 ligase for PDCD4 and triggers phosphorylation, ubiquitination and degradation of PDCD4. a MCF-7 cells were transfected with Flag-SKP2 and control plasmid for 48 h and harvested for IB. b MDA-MB-231 cells were transfected with sgSKP2 and control plasmid for 48 h and harvested for IB. c 293 T cells were transfected with Flag-SKP2 and control plasmid for 48 h. Cells were switched to fresh medium (10% FBS) containing AKT inhibitor MK-2206 (2.5 μM) 24 h or not post transfection and harvested for IB. d Flag–SKP2 proteins isolated 293 T cells transfected with Flag–SKP2 were incubated with ATP, E1, E2 along with PDCD4 proteins isolated from 293 T cells for in vitro ubiquitination assay. e In vivo ubiquitination assay from 293 T cells transfected with HA-Ubiquitin (HA-Ub) and indicated plasmids. f In vivo ubiquitination assay of 293 T cells transfected with HA-Ubiquitin (HA-Ub), HA-Ub K48R or HA-Ub K63R and indicated plasmids. g PDCD4 half-life is extended in SKP2−/− MEFs compared with SKP2 WT MEFs. Cells were switched to fresh medium (10% FBS) containing cycloheximide (CHX) 48 h post transfection for indicated time periods and harvested for IB. The band density was quantified