Fig. 1

SKP2 binds to PDCD4 and negatively regulates PDCD4 protein levels. a Lysates from 293 T cells stably transfected with vector or Myc-SKP2 were immunoprecipitated with Myc antibody and subjected to mass spectrometry analysis. PDCD4 was identified as a novel binding partner for SKP2. b, c 293 T cells were harvested for immunoprecipitation with SKP2 antibody (b) or PDCD4 antibody (c), followed by immunoblotting. d 35S-labeled in vitro translated SKP2 was used in binding reactions with beads coupled to the PDCD4 peptide K⁶⁵NSSRDSGRGDSVSD⁷⁹ or the phosphopeptide K⁶⁵NSpSRDSGRGDSVSD⁷⁹. Bound proteins were eluted and subjected to electrophoresis and autoradiography. e 293 T cells were transfected with indicated plasmids and harvested for immunoprecipitation assay. f SKP2 and PDCD4 expression of SKP2^−/− and WT MEF cells were determined by immunostaining (Scale bars, 10 um). g PDCD4 protein levels were increased in SKP2^−/− MEFs compared with WT MEFs. h PDCD4 and SKP2 protein expression levels in breast cancer cell lines were determined by IB and gray values were analyzed. Spearman rank correlation test was used to test of correlation between the SKP2 and PDCD4 protein expression levels, r = − 1.00, p < 0.01. i Overexpression of SKP2 decreased the levels of PDCD4 protein in MCF-7 cells: MCF-7 cells were transfected with SKP2 or vector control, followed by IB for protein expression. j SKP2 silencing increased the endogenous levels of PDCD4 protein in MDA-MB-231 cells: MDA-MB-231 cells were transfected with sgRNA and shRNA targeting SKP2, followed by IB for protein expression. k MCF-7 cells were transfected with SKP2 or vector control, followed by qRT-PCR for mRNA expression. l MDA-MB-231 cells were transfected with sgSKP2 or vector control, followed by qRT-PCR for mRNA expression. h, k, l Data represent the mean ± SEM of three independent experiments