Immunoblot analysis of Ras-signaling pathways in tumor cells, cell lines, or spheres. Hs578T, MCF7, MDA231 and breast cancer stem cells (BCSC) were plated in tumor spheroid media under adherent or non-adherent conditions. Tumor spheroids and adherent cells were collected at 96 hr, and lysed. MCF 10A (M1) and MCF 10C (M3) lysates were also prepared. The lysates were separated by electrophoresis and immunoblotted with antibodies against ERK, pEKR, AKT, pAKT, JNK, pJNK. Immunoblotting of α-tubulin serves as a loading control. For quantitation, the digital intensity of the bands was first normalized to α-tubulin in each lane, and then expressed relative to the signal for the MCF 10A (M1) cell line. Values are shown under each band.