Elucidating molecular mechanisms underlying brain metastasis-promoting function of Cdk5

To elucidate molecular mechanisms underlying brain metastasis-promoting function of Cdk5, two analyses were performed. In the first analysis, we screened for Cdk5 substrates in brain-seeking breast cancer cell lines 4T1.Br3 and T11.Br1, transfected with shRNA targeting Cdk5 mRNA (100% knockdown) or control. T11.Br1 and 4T1.Br3 cells (shCDK5 or shCtl) were cultured for 6 days in the presence of labelled arginine and lysine from the SILAC Protein Quantitation Kit. For the second analysis, we performed a quantitative label-free mass-spectrometry analysis of astrocytes’ conditioned media pre-educated by 4T1.Br3 cell-derived CM for 72 h. For the second analysis, we performed a quantitative label-free mass-spectrometry analysis of astrocytes’ conditioned media pre-educated by 4T1.Br3 cell-derived CM for 72 h.